Botanical Extracts and Compounds from Castanea Plants and Methods of Use

ABSTRACT

This disclosure relates to extracts from chestnut plants and compositions comprising compounds contained therein. In certain embodiments, the extracts are derived from the leaves of a  Castanea  plant. In certain embodiments, the disclosure relates to methods of treating or preventing bacterial infections, acne, and other related uses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to U.S. ProvisionalApplication Number 62/185,146 filed Jun. 26, 2015, hereby incorporatedby reference in its entirety for all purposes.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under R01AT007052awarded by NIH (NCCIH). The government has certain rights in theinvention.

BACKGROUND

Since the widespread introduction of antibiotics in the 1940s, the samestoryline has repeated itself over and over again: new antibiotic isintroduced and then resistant variants emerge and quickly spread,effectively limiting the utility and lifespan of the drug. From anevolutionary biology perspective, this is not surprising; indeed,resistant mutants are expected to arise when any lifeform with theability to rapidly reproduce and mutate is faced with a direct selectivepressure, especially when a single drug is used against a single target.Staphylococci are frequently the cause of hospital infections such asinfections from implanted medical devices. Many Staphylococci strainshave become resistant to many modern day antibiotics. Improved therapiesare needed.

One proposed strategy to overcome the problem of resistant variants isto indirectly attack bacteria by interfering with their means ofcommunication, also known as quorum sensing. Targeting microbialcommunication makes sense because bacteria coordinate many of theirvirulence and pathogenesis pathways through these systems. Quave et al.,report quorum sensing inhibitors of Staphylococcus aureus from botanicalextracts. Planta Med. 2011, 77(02):188-95.

Castanea sativa (chestnut) is a flowering plant in the family Fagaceaewhich can be found in Europe. See Braga et al., Nat Prod Res., 2015,29(1):1-18. Almeida et al. report in vivo skin irritation potential of aCastanea sativa (Chestnut) leaf extract. Basic & Clinical PharmacolToxicol, 2008, 103(5):461-7. See also Almeida et al. J PhotochemPhotobiol B: Biol, 2015, 144(0):28-34. Henry et al. report cosmeticcompositions containing an extract of leaves of the Castanea sativaplant and cosmetic treatments. U.S. Pat. No. 8,067,044 (2011).

Garo et al., report asiatic acid and corosolic acid enhance thesusceptibility of Pseudomonas aeruginosa biofilms to tobramycin.Antimicrob Agents Chemother, 2007, 51(5):1813-7. See also Rangasamy etal. South African J Botany, 2014, 93:198-203.

Wong et al. report aqueous methanolic extracts of Melastomamalabathricum L. exhibited antibacterial activity. Nat Prod Res,2012,26(7):609-18

Perioni et al. report a survey on the natural ingredients used in folkcosmetics, cosmeceuticals and remedies for healing skin diseases. JEthnopharmacol, 2004, 91(2-3):331-44.

References cited herein are not an admission of prior art.

SUMMARY

This disclosure relates to extracts from chestnut plants andcompositions comprising one or more compounds contained therein andrelated uses reported herein. In certain embodiments, the extracts arederived from the leaves of a Castanea plant such as Castanea sativa.

In certain embodiments, the disclosure relates to extracts comprising aleaf derived mixture of compounds from a Castanea plant wherein theextracting process comprises one or more of the following steps of:mixing a leaf with methanol under conditions such that leaf compoundsdissolves in the methanol and removing the methanol providing a methanolderived mixture of compounds; partitioning the methanol derived mixtureof compounds in hexane and water providing a water derived mixture ofcompounds; partitioning the water derived mixture of compounds by mixingthe water with ethyl acetate under conditions such that leaf compoundsdissolve in the ethyl acetate and removing the ethyl acetate providingan ethyl acetate derived mixture of compounds; and purifying the ethylacetate derived mixture of compounds by liquid chromatography throughsilica with a mobile phase comprising hexane and ethyl acetate; whereinthe mobile phase comprises increasing amounts of ethyl acetate, and amobile phase fraction is isolated comprising a leaf derived mixture ofcompounds which does not contain chlorogenic acid, ellagic acid,hyperoside, isoquercitrin, or rutin.

In certain embodiments, this disclosure relates to methods of treatingor preventing a bacterial infections or acne comprising administering toa subject in need thereof or contacting the skin of a subject in needthereof with a formula comprising an extract or one or more compounds inan extract as disclosed herein. In certain embodiments, the formula isadministered in combination with another antibiotic.

In certain embodiments, this disclosure relates to methods of treatingor preventing a toxin-mediated bacterial infection comprisingadministering an effective amount of an Castanea extract or compoundscontained therein to a subject in need thereof, including a subject atrisk of, exhibiting symptoms of, or diagnosed with a staphylococcalscalded skin syndrome (esp. in neonates), abscesses, necrotizingfasciitis, sepsis, or atopic dermatitis (eczema).

In certain embodiments, the subject is at risk of, exhibiting symptomsof, or diagnosed with toxic shock syndrome, scalded skin syndrome,abscesses, furuncles, cellulitis, folliculitis, bloodstream infections,medical device infections, pneumonia, osteomyelitis, staphylococcal foodpoisoning, skin and soft tissue infections, endocarditis, eczema, atopicdermatitis, psoriasis, impetigo, septic arthritis, brain abscess, burnwounds, venous ulcers, diabetic foot ulcers, surgical wounds,post-operation infections, carbuncles, meningitis, bacteremia,necrotizing pneumonia, or necrotizing fasciitis.

In certain embodiments, the disclosure contemplates the use of anextract or one or more compounds in an extract disclosed herein in atampon for the treatment or prevention of toxic shock syndrome.

In certain embodiments, the disclosure relates to a pharmaceutical orcosmetic formulation comprising an extract or one or more compounds inan extract disclosed herein and a pharmaceutically acceptable excipientor cosmetically acceptable excipient. In certain embodiments, thedisclosure relates to a liquid or gel formulation optionally furthercomprising an antibacterial agent, a topical steroid, ananti-inflammatory agent, a promoter of skin barrier function, a skinmoisturizer or combinations thereof. In certain embodiments theantibacterial agent is daptomycin, linezolid, vancomycin, nafcillin,cefazolin, dicloxacillin, clindamycin, rifampin,sulfamethoxale-trimethroprim (Bactrim), or botanical antibacterialagents, e.g., Melaleuca alternifolia tea tree oil.

In certain embodiments, the compound is in the form of an aqueoussolution further comprising a buffering agent, oil, phosphate buffer,sodium or potassium salt, a saccharide, polysaccharide, or solubilizingagent.

Uses as an injectable product (for intravenous, intramuscular,subcutaneous, intradermal injections, intraperitoneal, or otheradministration) are contemplated. In certain embodiments, the disclosurerelates to a pharmaceutical injectable formulation comprising an extractor one or more compounds in an extract disclosed herein and apharmaceutically acceptable excipient. In certain embodiments, thedisclosure relates to an injectable formulation optionally furthercomprising an antibacterial agent, a topical steroid, ananti-inflammatory agent, or combinations thereof. In certain embodimentsthe antibacterial agent is daptomycin, linezolid, vancomycin, nafcillin,cefazolin, dicloxacillin, clindamycin, rifampin,sulfamethoxale-trimethroprim (Bactrim), or botanical antibacterialagents, e.g., Melaleuca alternifolia tea tree oil.

In certain embodiments, the disclosure relates to a pharmaceuticalcomposition comprising an extract or one or more compounds in an extractdisclosed herein formulated with an enteric coating.

In certain embodiments, the disclosure relates to a solid or liquid soapor lotion comprising an extract or one or more compounds in an extractdisclosed herein and a fatty acid.

In certain embodiments, the disclosure relates to a medical devicecomprising a coating comprising an extract or one or more compounds inan extract disclosed herein.

In certain embodiments, the disclosure relates to a tampon or tamponfibers comprising an extract or one or more compounds in an extractdisclosed herein and an absorbent material.

In certain embodiments, the disclosure relates to a wound dressings orwound rinse comprising an extract or one or more compounds in an extractdisclosed herein wherein the wound dressing comprises an absorbent padand optionally an adhesive.

In certain embodiments, the disclosure relates to a disinfectant sprayor wipe formulation for surfaces and fomites, comprising an extract andone or one or more compounds in an extract disclosed herein wherein thespray or wipe comprises an extract or one or more compounds in anextract disclosed herein such as a formula including chlorine baseddisinfectants.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates a schematic of the Staphylococcus aureus accessorygene regulator system. The agr locus has been investigated in detail andis known to contain two divergent transcripts named RNAII and RNAIII TheRNAII transcript is an operon of four genes, agrBDCA, that encodefactors required to synthesize AlP and activate the regulatory cascade.Briefly, AgrD is the precursor peptide of AlP, AgrB is a membraneprotease involved in generating AlP, AgrC is a histidine kinase that isactivated by binding AlP, and AgrA is a response regulator that inducestranscription of both RNAII and RNAIII. The RNAIII transcript yields aregulatory RNA molecule that acts as the primary effector of the agrsystem by up-regulating extracellular virulence factors anddown-regulating cell surface proteins. The agr pathway is illustratedhere with potential target sites for 224C-F2.

FIG. 2A shows an extract isolation scheme. The bioassay-guidedfractionation scheme is illustrated, demonstrating the path from rawplant material to isolated, active natural products.

FIG. 2B shows overlapping HPLC chromatogram for the most activefractions corresponding to FIG. 2A illustrating how fractionationfunctions to increase the relative levels of active agents.

FIG. 3A shows data indicating chestnut leaf extracts inhibit for agr I,strain AH1677, in a non-biocide manner. S. aureus agr reporter strainswere treated with extracts 224, 224C, and 224C-F2 at a dose range of0.05-100m mL⁻¹. Bioactivity guided sequential fractionation resulted inincreased quenching of all 4 agr alleles in a manner independent ofgrowth inhibition. Optical density of the culture is represented bysolid black symbols; fluorescence in the agr reporters is indicated bythe open symbols. The IC₅₀ and IC₉₀ for quorum quenching impact of eachextract are reported in Table 3

FIG. 3B shows data for agr II, strain AH430, as in FIG. 3A.

FIG. 3C shows data for agr III, strain AH1747, as in FIG. 3A.

FIG. 4D shows data for agr IV, strain AH1872, as in FIG. 3A.

FIG. 4A shows data indicating 224C-F2 blocks MRSA exotoxin production.224C-F2 demonstrates a dose-dependent effect in inhibition ofde-formylated and formylated delta toxin, as illustrated in this HPLCchromatogram.

FIG. 4B show data on the quantification of delta-toxin confirmed thedose-dependent inhibitory activity of extracts, and the increasedactivity of the refined fraction 224C-F2 over 224 and 224C.

FIG. 4C shows data indicating extracts quench the hemolytic activity ofboth the S. aureus wild type and Δhla mutant, demonstrating that inaddition to preventing production of α-hemolysin hemolysin (responsiblefor the major share of hemolytic activity), that extracts also inhibitPSM production, responsible for the observable hemolytic activity in hlamutant strains.

FIG. 4D shows data for USA300 (Aspa) was exposed to increasing doses of224, 224C, 224C-F2, and vehicle control for 8 hrs. Western blot fora-hemolysin on supernatants demonstrated a dose-dependent decline inprotein levels.

FIG. 5 shows data where spent supernatants of S. aureus treated with224C-F2 exhibit diminished cytotoxic effects against humankeratinocytes. Supernatants were applied to HaCaT cells (20% v/v for 24hrs) to measure the lytic capacity (determined by LDH assay) of a fullsuite of S. aureus exotoxins. Supernatants from 224C-F2-treated cultureswere non-toxic to the mammalian cells, confirming inhibition of exotoxinproduction.

FIG. 6A shows data indicating 224C-F2 attenuates virulence without anydetectable resistance after 15 days of drug passaging. Cultures ofUSA500 isolate NRS385 (agr group I) were passaged for 15 consecutivedays in the presence of 16 μg mL⁻¹ of 224C-F2. The sum total peak areaof de-formylated and formylated delta toxin was quantified for the mockvehicle control (DMSO) and treated group. A significant difference(p<0.05) was evident for all treatment days.

FIG. 6B shows data indicating 224C-F2 inhibited delta-toxin productionover the length of the passaging experiment in the absence of growthinhibition.

FIG. 7 shows data indicating 224C-F2 is non-toxic to human keratinocytesand murine skin. Immortalized human keratinocytes (HaCaT cells) weretreated with up to 512 ₁.tg mL⁻¹ of extract fractions (24 hrs). The LD50for 224C-F2 could not be determined at this test range, indicating thatit is well above the active dose for quorum quenching activity(IC₅₀=1.56-25 μg mL⁻¹, depending on strain). Uninfected mice received anintradermal injection of 5 or 50 μg 224C-F2. No gross alterations inskin appearance were observed.

FIG. 8A shows data indicating 224C-F2 attenuates MRSA-induceddermatopathology in a murine model of skin and soft tissue infection.C5Bl/6 mice were intradermally injected with 1×10⁸ CFUs of LAC (USA 300isolate, AH1263) or its agr deletion mutant (AH1292). Mice received asingle dose of 224C-F2 (at 5 or 50 μg) or the vehicle control (DMSO) atthe time of infection. 224C-F2 attenuates dermatopathology with a singledose of either 5 or 50 μg.

FIG. 8B shows data indicating 224C-F2 reduces morbidity and mice do notlose weight.

FIG. 9A LC-FTMS ESl negative base peak chromatogram for 224C-F2. Peakscorrespond to data presented in Table 4. Putative structures arereported in FIG. 10.

FIG. 9B shows an expansion of the B segment provided in FIG. 9A.

FIG. 9C shows an expansion of the C segment provided in FIG. 9A.

FIG. 10 illustrates putative structures of ursene and oleanenederivatives found in the most active region of 224C-F2 (retention timeof 21-49 min) were determined following MS analysis and databasesearches. Compounds are listed by Peak number, corresponding to Table 4.Peak 31 was determined to be C₃₉H₅₉O₈ or C₃₈H₅₅O₉ with a relativeabundance of 0.34%. Putative structural matches include: (31a) escigenintetraacetate (6Cl); (31b) tetraacetate (7Cl,8Cl) 16α,21α-epoxy-olean-9(11)-ene-362 ,22β,24,28-tetrol; (31c) tetraacetateaescigenin; (31d) triacetate (8CI) cyclic16,22-acetal-olean-12-ene-3β,16α,21β,22α,28-pentol; (31e) triacetate(8CI) cyclic 22, 28-acetal-olean-12-ene-3β,16α,21β,22α,28-pentol. Peak32 was determined to be C₃₅H₅₉O₆ with a relative abundance of 0.30%.Putative structural matches include: (32a) stigmastane (FIGS. 11) and(32b) (3β,4β,16α,21β,22α)-16,21,22,23,28-pentamethoxy (9CI)olean-12-en-3-ol . Peak 42 was determined to be C₃₁H₄₉O₆ with a relativeabundance of 1.43%. Putative structural matches included (42) amirinicacid. Peak 52 was determined to be C₃₂H₅₁O₇ with a relative abundance of0.48%. Putative structural matches include: (52a) 21-acetateprotoescigenin, (52b) 16-acetate protoescigenin, (52c) 22-acetateprotoescigenin and (52d) 28-acetate protoescigenin. Peak 55 wasdetermined to be C₃₀H₄₈O₅, with a relative abundance of 4.11%. Putativestructural matches include: (55a)16,21-epoxy-(3β,4β,16α,21α,22β-olean-12-ene-3,22,24,28-tetrol (9CI);(55b) asiatic acid; (55c) arjunolic acid; (55d) isoescigenin. Peak 60was determined to be C₃₀H₄₈O₆, with a relative abundance of 6.80%.Putative structural matches include: (60a) camelliagenin E; (60b)brahmic acid; (60c) sericic acid; (60d) belleric acid; and (60e)2,3,23,24-tetrahydroxy-(2α,3β)-urs-12-en-28-oic acid. Peak 64 wasdetermined to be C₃₀H₄₅O₅, with a relative abundance of 2.91%. Theputative structural match is (64) ouillaic acid.

FIG. 11 shows putative structures of compounds other than pentacyclictriterpenes found in the most active region of 224C-F2 (retention timeof 21-49 min). Compounds are listed by peak number, corresponding toTable 4. Peak 32 was determined to be C₃₅H₅₉O₆ with a relative abundanceof 0.30%. Putative structural matches include: (32a) stigmastane and(32b) (3β,4β,16α, 21β,22α)-16,21,22,23,28-pentamethoxy (9CI)olean-12-en-3-ol (FIG. 10). Peak 33 was determined to be C₂₇H₂₃O₁₈ witha relative abundance of 0.16%. Putative structural matches include:(33a) 1,3 ,6-tri-O-galloylglucose; (33b) 1,2,6-tri-galloyl-β-D-glucose;(33c) 1,2,3 -tri-O-galloylglucose; (33d) 1,2,3-tri-O-galloyl-β-D-glucopyranose; (33e)2′,3,5-tri-O-galloyl-D-hamamelose; (33f) 2-C-[[(3,4,5-trihydroxybenzoyl)oxy]methyl]-1,5-bi s(3 ,4,5-trihydroxybenzoate)D-Ribofuranose; (33g) kurigalin; (33h) 3,4,6-tri-O-galloyl-D-glucose.Peak 34 was determined to be C₃₉H₃₁O₁₅ with a relative abundance of0.65%. Putative structural matches include: (34) castanoside B. Peak 39was determined to be C₁₇H₁₁O₈ or C₂₀H₁₁O₄N₂ with a relative abundance of0.72%. Putative structural matches include: (39a)3,4,3′-tri-O-methylellagic acid and (39b) 3,3′,4′-tri-O-methylellagicacid. Peak 44 was determined to be C₃₄H₂₉O₁₅ with a relative abundanceof 0.26%. Putative structural matches included (44) norbadione A.

FIG. 12 shows scheme including sub fractions of 224C F2c semi-prep RPHPLC after flash chromatography.

FIG. 13 shows RP HPLC and fractions.

FIG. 14 shows LC-FTMS ESI negative of 224C F2c SF7.

FIG. 15 shows LC-FTMS ESI negative of 224C F2c SF11.

FIG. 16 shows Agr I activity and growth following treatment withsubfractions 224C-F2c-SF7 and 224C-F2c-SF 11 on S. aureus growth(measured in optical density, OD), accompanied by inhibition of agr Ifunction (measured in fluorescent units, RFU) in a dose dependentfashion.

DETAILED DISCUSSION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. In this specification andin the claims that follow, reference will be made to a number of termsthat shall be defined to have the following meanings unless a contraryintention is apparent.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

As used herein, “subject” refers to any animal, preferably a humanpatient, livestock, or domestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity is reduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, “relative abundance” refers to amount determined fromelectrospray ionization mass spectrometry. Mass spectrometry is ananalytical technique that can provide both qualitative (structure) andquantitative (molecular mass or concentration) information on analytemolecules after their conversion to ions. The molecules of interest arefirst introduced into the ionization source of the mass spectrometer,where they are first ionized to acquire positive or negative charges.The ions then travel through the mass analyzer and arrive at differentparts of the detector according to their mass/charge (m/z) ratio. Afterthe ions make contact with the detector, useable signals are generatedand recorded by a computer system. The computer displays the signalsgraphically as a mass spectrum showing the relative abundance of thesignals according to their m/z ratio.

Chestnut Leaf Extracts Block Staphylococcus aureus Virulence andPathogenesis

Quorum quenching activity has been discovered in the natural productsextracted from Castanea sativa leaves. The extract is able to attenuatevirulence by quenching S. aureus agr-mediated quorum sensing,effectively blocking production of harmful exotoxins at sub-inhibitoryconcentrations for growth. Experiments indicate a lack of cytotoxicityto human skin cells, lack of growth inhibitory activity against thenormal skin microflora, lack of resistance development, and efficacy ina skin abscess animal model.

Staphylococcus aureus is an abundant, opportunistic pathogen that is thecausative agent of numerous infections. Due to its prevalence as aleading cause of healthcare-associated infection, and its highlymultidrug resistant nature, S. aureus is a serious threat. It colonizesthe nasal passages of approximately 30% of the healthy adult population.S. aureus infections initiate through trauma to the skin or mucosallayer and then progress through an invasive or toxin-mediated process.The prevalence of these infections has increased due to higher rates ofimmunosuppressive conditions, greater use of surgical implants, anddramatic increases in antibiotic resistance.

S. aureus produces an extensive array of enzymes, hemolysins, and toxinsthat are important to its ability to spread through tissues and causedisease. These virulence factors serve a wide scope of purposes in theinfection process, including disruption of the epithelial barrier,inhibition of opsonization by antibody and complement, neutrophilcytolysis, interference with neutrophil chemotaxis, and inactivation ofantimicrobial peptides. The expression of all of these invasive factorsis controlled by cell-density quorum sensing using the autoinducingpeptide (AIP) molecule. Like other quorum-sensing signals, AIPaccumulates outside the cell until it reaches a critical concentrationand then binds to a surface receptor called AgrC, initiating aregulatory cascade. Since AIP controls the expression of accessoryfactors for S. aureus, this regulatory system has been named theaccessory gene regulator (agr), and the majority of the proteinsnecessary for this quorum-sensing system to function are encoded in theagr chromosomal locus. Applying inhibitors to quench this communicationsystem to attenuate pathogenicity and virulence lies at the core of thequorum quenching approach.

Agr plays a key role in S. aureus pathogenesis. For example, skin andsoft tissue infections are the most common type of infection caused byS. aureus. These range from minor inflammatory conditions to moreinvasive infection, and most of these cases are associated with theformation of abscesses, the hallmark of a S. aureus infection. The bulkof the phenotype is due to agr-dependent secreted virulence factors.Interference with the agr system through the use of competing AIPs orAIP-sequestering antibodies decreased abscess formation. These findingsprovide direct support for the notion that agr-targeted therapies couldbe an option for the development of skin infection treatments. Lookingat other types of infections, agr mutants also display attenuatedvirulence in mice in the establishment of pneumonia and mortality, andin a systemic bloodstream infection model.

Given the importance of the agr system in pathogenesis, it has becomethe target of a number of chemical anti-virulence approaches. With theextracellular exposure of the AgrC receptor, chemists have developedreceptor antagonists that successfully inhibit the system in vitro.

Castanea sativa leaves were identify as a potential source newanti-infective agents. Through design of a bioactivity-guidedfractionation strategy based on limited growth-impact coupled to quorumsensing inhibition, a highly efficacious botanical composition withuniversal quenching activity was created for all agr alleles.

Reported herein are quorum quenching effects of a botanical compositionrich in ursene and oleanene derivatives (FIG. 10) against S. aureus.Additional compounds identified in the most active region (at <1%relative abundance each) included putative gallotannins, which share atri-galloyl structure with varying core sugars, and a putativeellagitannin.

Safety studies in both human keratocytes (HaCaT cells) and murine skin(FIG. 6) have confirmed that this version of chestnut leaf extract(224C-F2) can be considered safe for topical applications based on itslack of cytotoxic and irritant effects.

Several layers of evidence in support of the efficacy of C. sativa leafextracts in blocking S. aureus virulence have been presented.Specifically, chestnut leaf extracts are effective in blockingproduction of the translational products of RNAIII, including a numberof exotoxins. Overall virulence was quenched as demonstrated by the lackof cytotoxic effects elicited by supernatants of cultures treated withthe extract. Importantly, using an in vivo model, efficacy inattenuating dermonecrosis was demonstrated, even in the absence ofadjuvant antibiotics.

This inhibition of virulence and pathogenesis was accomplished withoutposing growth inhibitory pressures on not only S. aureus, but also apanel of common members of the human cutaneous microbiome. A robust skinmicroflora is critical to skin barrier health and prevention of diseaseonset. The majority of the bacterial cutaneous microbiome is representedby Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes. Muchlike cases of dysbiosis in gut microflora, broad-spectrum activityagainst the skin microflora also holds the potential for fostering anenvironment amenable to the proliferation of pathogenic bacteria. Thepresence of commensals, like Staphylococcus epidermidis, is essential tothe state of host innate immunity. Thus, it is noteworthy that 224C-F2specifically blocks S. aureus virulence without adding selectivepressures on major representatives of the cutaneous microbiome.

Multiple lines of evidence suggest that components within 224C-F2directly target the core machinery of the agr system, such as theobservation of agr P3 promoter reduction (FIG. 3) and reduced levels ofδ-toxin production (FIG. 4), which is encoded within RNAIII transcriptregulated by P3. If 224C-F2 only targeted downstream factors regulatedby quorum sensing, such as α-hemolysin, inhibition of agr P3 or δ-toxinproduction would not have been expected. Potential targets within theagr system include inhibition of AIP docking with AgrC, prevention ofAIP production through AgrB, or reduction of AgrA activation (FIG. 1).

Following 15 days of sequential passaging with 224C-F2 in vitro, noresistance was detected. Thus, it is contemplated that 224C-F2 andcompounds contained therein are a therapeutic option due to its abilityto specifically target and quench S. aureus virulence. Importantly, thiscomposition was non-toxic to human keratinocytes and no dermatopathologywas noted upon administration to murine skin. Moreover, the compositiondid not inhibit growth of the normal skin microflora, suggesting thatits disruptive action on the cutaneous microbiome would be minimal.

One major benefit of using virulence inhibitors with a classicalantibiotic is the potential for increased antibiotic efficacy. Byblocking the toxins responsible for immune response damage, theantibiotics and immune system can work more in concert to eliminate thebacteria.

Other topical formulations for skin flares (i.e. for atopic dermatitisor other infections related to a disrupted skin barrier) that may becombined with the anti-virulence drug include: topical steroids,anti-inflammatory agents, and promoters of skin barrier function or skinmoisturizers such as ceramide, glycerin, colloidal oatmeal.

In certain embodiments the disclosure contemplates that an extract orone or more compounds in an extract disclosed herein may be used as avirulence inhibitor applications optionally in combination with otherantibacterial agents for prevention of disease onset and treatment suchas in medical device coatings (medical implants and tools, IVcatheters), wound dressings (embedded in gauze bandages), wound rinses(i.e. surgical rinses), wound-vacuum systems, whole body baths (e.g., incombo with bleach baths for treatment of skin flares for atopicdermatitis/eczema), soaps, personal care products (body washes, lotions,soaps) for high risk patients or for populations with high risk ofexposure (e.g. athletes using common sports equipment in gym) human andveterinary applications (e.g. anti-infectives for companion animals,race horses, etc.)

Methods of Use

In certain embodiments, this disclosure relates to methods of treatingor preventing a bacterial infections comprising administering orcontacting a formula comprising an extract or one or more compounds inan extract as disclosed herein to a subject in need thereof. In certainembodiments, the formula is administered in combination with anotherantibiotic agent.

In further embodiments, the subject is co-administered with anantibiotic selected from the group comprising of sulfonamides,diaminopyrimidines, quinolones, beta-lactam antibiotics, cephalosporins,tetracyclines, notribenzene derivatives, aminoglycosides, macrolideantibiotics, polypeptide antibiotics, nitrofuran derivatives,nitroimidazoles, nicotinin acid derivatives, polyene antibiotics,imidazole derivatives or glycopeptide, cyclic lipopeptides,glycylcyclines and oxazolidinones. In further embodiments, theseantibiotics include but are not limited to sulphadiazine,sulfones—[dapsone (DDS) and paraaminosalicyclic (PAS)], sulfanilamide,sulfamethizole, sulfamethoxazole, sulfapyridine, trimethoprim,pyrimethamine, nalidixic acids, norfloxacin, ciproflaxin, cinoxacin,enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin,lomefloxacin, moxifloxacin, ofloxacin, pefloxacin, sparfloxacin,trovafloxacin, penicillins (amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, hetacillin,oxacillin, mezlocillin, penicillin G, penicillin V, piperacillin),cephalosporins (cefacetrile, cefadroxil, cefalexin, cefaloglycin,cefalonium, cefaloridin, cefalotin, cefapirin, cefatrizine, cefazaflur,cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor,cefonicid, ceforanide, cefprozil, cefuroxime, cefuzonam, cefmetazole,cefoteta, cefoxitin, cefcapene, cefdaloxime, cefdinir, cefditoren,cefetamet, cefixime, cefmenoxime, cefodizime, cefoperazone, cefotaxime,cefotiam, cefpimizolecefpiramide, cefpodoxime, cefteram, ceftibuten,ceftiofur, ceftiolen, ceftizoxime, ceftriaxone, cefoperazone,ceftazidime, cefepime), moxolactam, carbapenems (imipenem, ertapenem,meropenem) monobactams (aztreonam)oxytetracycline, chlortetracycline,clomocycline, demeclocycline, tetracycline, doxycycline, lymecycline,meclocycline, methacycline, minocycline, rolitetracycline,chloramphenicol, amikacin, gentamicin, framycetin, kanamycin, neomycin,netilmicin, streptomycin, tobramycin, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, telithromycin, colistin,bacitracin, tyrothricin, notrifurantoin, furazolidone, metronidazole,tinidazole, isoniazid, pyrazinamide, ethionamide, nystatin,amphotericin-B, hamycin, miconazole, clotrimazole, ketoconazole,fluconazole, rifampacin, lincomycin, clindamycin, spectinomycin,fosfomycin, loracarbef, polymyxin B, polymyxin B Sulfate, procain,ramoplanin, teicoplanin, vancomycin, and/or nitrofurantoin.

In certain embodiments, this disclosure relates to methods of treatingor preventing a toxin-mediated bacterial infection comprisingadministering an effective amount of a Castanea extract or compoundscontained therein to a subject in need thereof, including a subject atrisk of, exhibiting symptoms of, or diagnosed with a staphylococcalscalded skin syndrome (esp. in neonates), abscesses, necrotizingfasciitis, sepsis, atopic dermatitis (eczema) and more.

In certain embodiments, the subject is at risk of, exhibiting symptomsof, or diagnosed with toxic shock syndrome, scalded skin syndrome,abscesses, furuncles, cellulitis, folliculitis, bloodstream infections,medical device infections, pneumonia, osteomyelitis, staphylococcal foodpoisoning, skin and soft tissue infections, endocarditis, eczema, atopicdermatitis, psoriasis, impetigo, septic arthritis, brain abscess, burnwounds, venous ulcers, diabetic foot ulcers, surgical wounds,post-operation infections, carbuncles, meningitis, bacteremia,necrotizing pneumonia, or necrotizing fasciitis.

In certain embodiments, the disclosure contemplates methods ofpreventing bacterial infections by applying extracts or one or morecompounds in extracts disclosed herein in a tampon for preventionagainst adverse effects associated with vaginal area infections andpossibly bladder infections, e.g., toxic shock syndrome. As used hereina “tampon” refers to device containing an absorbent material, configuredto be inserted into a vagina to absorb menstrual flow and typicallyexpand during use, typically in the shape of a cylinder. Tampons mayexpand axially (increase in length), while digital tampons will expandradially (increase in diameter). Most tampons have a cord or string forremoval. Typical tampon materials include cloth, fibers, cotton, orrayon, or a blend of rayon and cotton.

Bacterial toxins may cause toxic shock syndrome (TSS). Enterotoxin typeB or TS ST-1 of Staphylococcus aureus are believed to cause TSS.Streptococcal TSS is sometimes referred to as toxic shock-like syndrome(TSLS) or streptococcal toxic shock syndrome (STSS). CDC criteria fordiagnosing staphylococcal toxic shock syndrome is based on 1) a bodytemperature of greater than 38.9° C. (102.02° F.) 2) a Systolic bloodpressure of greater than 90 mmHg 3) diffuse macular erythroderma 4)desquamation (especially of the palms and soles) 1-2 weeks after onset5) involvement of three or more organ systems: gastrointestinal(vomiting, diarrhea), muscular: severe myalgia or creatine phosphokinaselevel at least twice the upper limit of normal for laboratory, mucousmembrane hyperemia (vaginal, oral, conjunctival), kidney failure (serumcreatinine>2 times normal), liver inflammation (bilirubin, AST, or ALT>2times normal), low platelet count (platelet count<100,000/mm³), Centralnervous system involvement (confusion without any focal neurologicalfindings) and 6) Negative results of: blood, throat, and CSF culturesfor other bacteria (besides S. aureus) negative serology for Rickettsiainfection, leptospirosis, and measles. Cases are classified as probableis five of the six criteria above are met.

In certain embodiments, the disclosure contemplates methods ofpreventing general transmission of bacterial through use of extracts andone or more compounds in an extract disclosed herein as a general agentformulated into a spray or wipe product, paper or fiber based cloth. Forexample, one can use such a product to treat athletic equipment(football pads, bench presses, gym surfaces), where invasive toxinmediated staph often lurks and causes infections in healthy peoplethrough toxin production.

In certain embodiments, the disclosure relates to methods of treatingacne comprising administering an effective amount of a compositioncomprising an extract or one or more compounds in an extract asdisclosed herein to a subject at risk of, exhibiting symptoms of, ordiagnosed with acne, blackheads, papules, pustules or nodules. Incertain embodiments, the subject is undergoing puberty, between 10 and20 years of age. In certain embodiments, the subject is a female, andthe composition is administered within seven days of the beginning of amenstrual cycle. Administration may be by topical application throughhand or by spray of a liquid or lotion containing an extract or one ormore compounds in an extract disclosed herein.

Extracts and Compounds

In certain embodiments, an extract is made by the process of extractinga mixture of compounds from the leaves, roots, bark, stem, or branchesof a Castanea plant e.g., Castanea sativa. Other contemplated plantsinclude: Castanea acuminatissima, Castanea alabamensis, Castaneaalnifolia, Castanea americana, Castanea argentea, Castanea argyrophylla,Castanea arkansana, Castanea armata, Castanea ashei, Castaneablaringhemii, Castanea bodinieri, Castanea brevicuspis, Castaneabungeana, Castanea burbankii, Castanea buruana, Castanea californica,Castanea Castanea, Castanea castanicarpa, Castanea castenea var.pubinervis, Castanea chincapin, Castanea chinensis, Castaneachrysophylla, Castanea concinna, Castanea cooperta, Castanea costata,Castanea coudersii, Castanea crenata, Castanea davidii, Castaneadentata, Castanea diversifolia, Castanea dovaricata, Castanea duclouxii,Castanea echidnocarpa, Castanea edonii, Castanea edwii, Castaneaendicottii, Castanea eonii, Castanea fagus, Castanea falconeri, Castaneafargesii, Castanea fauriei, Castanea fleetii, Castanea floridana,Castanea formosana, Castanea furfurella, Castanea glomerata, Castaneahenryi, Castanea henryi, Castanea hupehensis, Castanea hystrix,Castanea, Castanea inermis, Castanea japonica, Castanea javanica,Castanea kusakuri, Castanea lanceifolia, Castanea latifolia, Castaneamargaretta, Castanea martabanica, Castanea microcarpa, Castaneamollissima, Castanea montana, Castanea morrisii, Castanea nana, Castaneaneglecta, Castanea ozarkensis, Castanea paucispina, Castaneaphansipanensis, Castanea prolifera, Castanea pubinervis, Castaneapulchella, Castanea pumila, Castanea purpurella, Castanea regia,Castanea rhamnifolia, Castanea rockii, Castanea roxburghii, Castaneaseguinii, Castanea sempervirens, Castanea sessilifolia, Castaneasinensis, Castanea sloanea, Castanea spectabilis, Castanea sphaeroarpa,Castanea sphaerocarpa, Castanea stricta, Castanea sumatrana, Castaneatribuloides, Castanea tungurrut, Castanea vesca, Castanea vilmoriniana,Castanea vulgaris, Castanea wattii and hybrids thereof.

In certain embodiments, the extracting process comprises the step ofmixing the leaf from the plant with a polar solvent, such as a liquidcomprising methanol, ethanol, ethyl acetate, acetonitrile, acetone,methylene chloride or chloroform, under conditions such that a mixtureof compounds in the leaf dissolves in the solvent. In certainembodiments, the process further comprises the step of removing thesolvent by evaporation from the mixture of compounds. In certainembodiments, the process further comprises the step of purifying themixture of compounds by liquid chromatography through a solid absorbent,e.g., wherein the solid absorbent comprises silica gel or alumina.

In certain embodiments, the disclosure relates to extracts comprising aleaf derived mixture of compounds from a Castanea plant wherein theextracting process comprises the steps of: mixing a leaf with methanolunder conditions such that leaf compounds dissolves in the methanol andremoving the methanol providing a methanol derived mixture of compounds;partitioning the methanol derived mixture of compounds in hexane andwater providing a water derived mixture of compounds; partitioning thewater derived mixture of compounds by mixing the water with ethylacetate under conditions such that leaf compounds dissolve in the ethylacetate and removing the ethyl acetate providing an ethyl acetatederived mixture of compounds; and purifying the ethyl acetate derivedmixture of compounds by liquid chromatography through silica with amobile phase comprising hexane and ethylene acetate; wherein the mobilephase comprises increasing amounts of ethyl acetate, and a mobile phasefraction is isolated comprising a leaf derived mixture of compoundswhich does not contain chlorogenic acid, ellagic acid, hyperoside,isoquercitrin, or rutin.

Chromatography refers to the separation of a mixture of compoundsdissolved in a fluid called the mobile phase, which carries thecompounds through a structure holding another material called thestationary phase. The various compounds or components of the mixturetravel at different speeds, causing them to separate. The separation isbased on differential partitioning between the mobile and stationaryphases. Subtle differences in a partition coefficient of each compoundresult in differential retention on the stationary phase and thuschanging the separation.

In normal-phase chromatography, the stationary phase is polar. Inreversed phase, the stationary phase is nonpolar. Typical stationaryphases for normal-phase chromatography are silica or organic moietieswith cyano and amino functional groups. For reversed phase, alkylhydrocarbons are the preferred stationary phase. Examples are solidsupports containing a surface conjugated with a hydrocarbon chain, e.g.,octadecyl (C18), octyl (C8), and butyl (C4).

In normal-phase chromatography, the least polar compounds elute firstand the most polar compounds elute last. The mobile phase typicallyconsists of a nonpolar solvent such as hexane or heptane mixed with aslightly more polar solvent such as isopropanol, ethyl acetate orchloroform. Retention to the stationary phase decreases as the amount ofpolar solvent in the mobile phase increases. In reversed phasechromatography, the most polar compounds elute first with the mostnonpolar compounds eluting last. The mobile phase is generally a binarymixture of water and a miscible polar organic solvent like methanol,acetonitrile or THF.

In certain embodiments, methods of extraction comprise mixing leaves ofa Castanea plant with an water miscible carbon containing solvent, e.g.,such as a protic solvent, an alcohol, methanol, ethanol, 1-propanol,2-propanol, tetrahydrofuran, acetone, acetic acid, 1,4-dioxane ormixture providing a concentrate with a mixture of compounds andsubstantially removing the solvent from the concentrate, purifying thesolvent derived concentrate to less than 5%, 1%, or 0.5% by weight ofthe solvent used in the extraction, e.g., evaporating the protic solventand/or optionally in combination with mixing the concentrate with water,sonicating the water, freezing the water to provide ice, and removingthe ice by sublimation (e.g. in a vacuum of low pressure) wherein saidpurification methods may be repeated in combination. In certainembodiments, the method further comprises suspending the solvent derivedconcentrate in water and optionally extract impurities in a hydrocarbonsolvent such as cyclohexane, heptane, hexane, pentane,2,2,4-trimethylpentane, separating the hydrocarbon from the waterproviding a water layer. In certain embodiments, the method furthercomprises mixing the water layer with a solvent that is immiscible inwater (polar and/or aprotic), e.g., such as ethyl acetate, diethylether, methyl tertbutyl ether, toluene, methylene chloride, carbontetrachloride, 1,2-di chl oroethant, and/or chloroform, and purifyingthe solvent to provide a second solvent derived concentrate. In furtherembodiments, the second derived concentrate is purified one or moretimes by liquid chromatography, e.g., normal phase chromatography.Typically the solid absorbent is polar such as silica. In certainembodiments, the extract is a portion isolated after the column solventis more than 50% ethyl acetate in hexane.

In certain embodiments, the mixture of compounds comprises components ofthe following formulas and relative abundance C₅₇H₂₄O₂ (2.67%), C₂₇H₅₀O₆(2.65%), C₃₁H₅₀O₆ (1.43%), C₃₀H₄₆O₇ (1.86%), and two compoundsC₅₇H₂₃O₂N₃ (1.64% and 3.13%, respectively), two compounds C₅₉H₂₅O₃ (1.45and 1.07%, respectively), C₄₁H₃₃O₁₆ (1.20%) and C₃₀H₄₇O₅ (5.96%).

In certain embodiments, the mixture of compounds comprises componentshaving the following formulas and relative abundance, C₃₉H₅₉O₈ orC₃₈H₅₅O_(9,) or mixture, with a relative abundance of 0.1 to 0.9%, e.g.0.3%; C₃₅H₅₉O₆ with a relative abundance of 0.1 to 0.9%, e.g., 0.3%;C₃₁H₄₉O₆ with a relative abundance of 1.0 to 2.0%, e.g., 1.4%; C₃₂H₅₁O₇with a relative abundance of 0.1 to 0.9%, e.g., 0.5%; C₃₀H₄₈O₆ with arelative abundance of 3.0 to 5.0%, e.g., 4.1%; C3oH4806 with a relativeabundance of 6.0 to 8.0%, e.g., 6.8%; and C₃₀H₄₅O₅ with a relativeabundance of 2.0 to 4.0%, e.g., 2.9%. Typically the mixture furthercomprises additional components of different compounds having thefollowing formula and relative abundance, C₃₅H₅₉O₆ with a relativeabundance of 0.2 to 0.4, e.g., 0.30%; C₂₇H₂₃O₁₈ with a relativeabundance of 0.05 to 0.30, e.g., 0.16%; C₃₉H₃₁O₁₅ with a relativeabundance of 0.50 to 0.80, e.g., 0.65%; C₁₇H₁₁O₈ or C₂₀H₁₁O₄N_(2,) ormixture, with a relative abundance of 0.5 to 1.0, e.g., 0.7%; andC₃₄H₂₉O₁₅ with a relative abundance of 0.2 to 0.4%, e.g., 0.3%.

In certain embodiment, the extract comprises at mixture comprises atleast one component from each of the following groups a) to g): a) acompound selected from escigenin tetraacetate (6CI); tetraacetate(7CI,8CI) 16α,21α-epoxy-olean-9(11)-ene-3β,22β,24,28-tetrol;tetraacetate aescigenin; triacetate (8CI) cyclic16,22-acetal-olean-12-ene-3β,16α,21β,22α,28-pentol; and triacetate (8CI)cyclic 22,28-acetal-olean-12-ene-3β,16α,21β,22α,28-pentol, or mixture oftwo, three, four or all; b) a compound selected from stigmastane and(3β,4β,16α,21β,22α)-16,21,22,23,28-pentamethoxy (9CI) olean-12-en-3-ol,or mixture; c) amirinic acid; d) a compound selected from 21-acetateprotoescigenin, 16-acetate protoescigenin, 22-acetate protoescigenin and28-acetate protoescigenin, or mixture of two, three, four or all;

e) a compound selected from16,21-epoxy-(3β,4β,16α,21α,22β)-olean-12-ene-3,22,24,28-tetrol (9CI);asiatic acid; arjunolic acid; and isoescigenin, or mixture of two,three, or all;

f) a compound selected from camelliagenin E; brahmic acid; sericic acid;belleric acid; and 2,3,23,24-tetrahydroxy-(2α,3β)-urs-12-en-28-oic acid,or mixture of two, three, four or all; and

g) ouillaic acid. Typically, the mixture further comprises at least onecomponent from each of the following groups a) to d): a) a compoundselected from stigmastane and(3β,4β,16α,21β,22α)-16,21,22,23,28-pentamethoxy (9CI) olean-12-en-3-olor a mixture; b) a compound selected from 1,3,6-tri-O-galloylglucose;1,2,6-tri-galloyl-β-D-glucose; 1,2,3-tri-O-galloylglucose;1,2,3-tri-O-galloyl-β-D-glucopyranose;2′,3,5-tri-O-galloyl-D-hamamelose;2-C-[[(3,4,5-trihydroxybenzoyl)oxy]methyl]-1,5-bis(3,4,5-trihydroxybenzoate)D-ribofuranose; kurigalin; and 3,4,6-tri-O-galloyl-D-glucose, or mixtureof two, three, four, five, six, seven, or all; c) castanoside B; and d)a compound selected from 3,4,3′-tri-O-methylellagic acid and3,3′,4′-tri-O-methylellagic acid, or a mixture.

Pharmaceutical Formulation

In certain embodiments, the disclosure relates to a pharmaceuticalformulation comprising an extract or one or more compounds in an extractdisclosed herein and a pharmaceutically acceptable excipient oradditive. In certain embodiments, the disclosure relates to a lotion,liquid, or gel formulation optionally further comprising an antibioticagent, a topical steroid, an anti-inflammatory agent, a promoter of skinbarrier function, a skin moisturizer or combinations thereof.

Examples of antibiotics include but are not limited to sulphadiazine,sulfones—[dapsone (DDS) and paraaminosalicyclic (PAS)], sulfanilamide,sulfamethizole, sulfamethoxazole, sulfapyridine, trimethoprim,pyrimethamine, nalidixic acids, norfloxacin, ciproflaxin, cinoxacin,enoxacin, gatifloxacin, gemifloxacin, grepafloxacin, levofloxacin,lomefloxacin, moxifloxacin, ofloxacin, pefloxacin, sparfloxacin,trovafloxacin, penicillins (amoxicillin, ampicillin, azlocillin,carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, hetacillin,oxacillin, mezlocillin, penicillin G, penicillin V, piperacillin),cephalosporins (cefacetrile, cefadroxil, cefalexin, cefaloglycin,cefalonium, cefaloridin, cefalotin, cefapirin, cefatrizine, cefazaflur,cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor,cefonicid, ceforanide, cefprozil, cefuroxime, cefuzonam, cefmetazole,cefoteta, cefoxitin, cefcapene, cefdaloxime, cefdinir, cefditoren,cefetamet, cefixime, cefmenoxime, cefodizime, cefoperazone, cefotaxime,cefotiam, cefpimizolecefpiramide, cefpodoxime, cefteram, ceftibuten,ceftiofur, ceftiolen, ceftizoxime, ceftriaxone, cefoperazone,ceftazidime, cefepime), moxolactam, carbapenems (imipenem, ertapenem,meropenem) monobactams (aztreonam)oxytetracycline, chlortetracycline,clomocycline, demeclocycline, tetracycline, doxycycline, lymecycline,meclocycline, methacycline, minocycline, rolitetracycline,chloramphenicol, amikacin, gentamicin, framycetin, kanamycin, neomycin,netilmicin, streptomycin, tobramycin, azithromycin, clarithromycin,dirithromycin, erythromycin, roxithromycin, telithromycin, colistin,bacitracin, tyrothricin, notrifurantoin, furazolidone, metronidazole,tinidazole, isoniazid, pyrazinamide, ethionamide, nystatin,amphotericin-B, hamycin, miconazole, clotrimazole, ketoconazole,fluconazole, rifampacin, lincomycin, clindamycin, spectinomycin,fosfomycin, loracarbef, polymyxin B, polymyxin B Sulfate, procain,ramoplanin, teicoplanin, vancomycin, and/or nitrofurantoin.

Examples of steroids include hydrocortisone, hydrocortisone valerate,hydrocortisone 17-butyrate, mometasone, mometasone furoate, halobetasolpropionate, desonide, desoximetasone, fluocinolone acetonide,alclometasone dipropionate, flurandrenolide, fluticasone propionate,diflucortolone, diflucortolone valerate, diflorasone diacetate,clobetasol, clobetasone butyrate, clobetasol propionate, betamethasonedipropionate, betamethasone valerate, beclomethasone, budesonide,flunisolide, fluocinonide, triamcinolone, triamcinolone acetonide,methylprednisolone, methylprednisolone aceponate, prednicarbate,prednisolone, and prednisone and alternate salts thereof. Examples ofcontemplated anti-inflammatory agents are aspirin, celecoxib,diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen,naproxen, oxaprozin, and piroxicam.

In certain embodiments, the disclosure relates to a pharmaceuticalcomposition comprising an extract or one or more compounds in an extractdisclosed herein formulated with an enteric coating. In certainembodiments, the disclosure relates to a pharmaceutical formulation ofan extract or one or more compounds in an extract disclosed herein whichprotect the compositions from the acidity and enzymatic action ofgastric secretions. In certain embodiments, the pharmaceuticalformulations contain an extract or one or more compounds in an extractdisclosed herein in a composition with an enteric coating along withanother pharmaceutically acceptable vehicle. In certain embodiments,compositions comprising an extract or one or more compounds in anextract disclosed herein may be directly-compressible withoutexcipients, into a tablet of pharmaceutically acceptable hardness, e.g.,compressed into a tablet, optionally with a lubricant, such as but notlimited to magnesium stearate, and enteric coated. In anotherembodiment, the pharmaceutical compositions containing an extract or oneor more compounds in an extract disclosed herein alternatively includeone or more substances that either neutralize stomach acid and/orenzymes or are active to prevent secretion of stomach acid.

The pharmaceutical composition can be formulated for oral administrationas, for example but not limited to, drug powders, crystals, granules,small particles (which include particles sized on the order ofmicrometers, such as microspheres and microcapsules), particles (whichinclude particles sized on the order of millimeters), beads, microbeads,pellets, pills, microtablets, compressed tablets or tablet triturates,molded tablets or tablet triturates, and in capsules, which are eitherhard or soft and contain the composition as a powder, particle, bead,solution or suspension. The pharmaceutical composition can also beformulated for oral administration as a solution or suspension in anaqueous liquid, as a liquid incorporated into a gel capsule or as anyother convenient formulation for administration, or for rectaladministration, as a suppository, enema or other convenient form.

The injectable solutions or suspensions may be formulated according toknown art, using suitable non-toxic, parenterally-acceptable diluents orsolvents, such as mannitol, 1,3-butanediol, water, Ringer's solution orisotonic sodium chloride solution, or suitable dispersing or wetting andsuspending agents, such as sterile, bland, fixed oils, includingsynthetic mono- or diglycerides, and fatty acids, including oleic acid.

Suitably, the pharmaceutical composition of the disclosure comprises acarrier and/or diluent appropriate for its delivering by injection to ahuman or animal organism. Such carrier and/or diluent is non-toxic atthe dosage and concentration employed. It is selected from those usuallyemployed to formulate compositions for parental administration in eitherunit dosage or multi-dose form or for direct infusion by continuous orperiodic infusion. It is typically isotonic, hypotonic or weaklyhypertonic and has a relatively low ionic strength, such as provided bysugars, polyalcohols and isotonic saline solutions. Representativeexamples include sterile water, physiological saline (e.g. sodiumchloride), bacteriostatic water, Ringer's solution, glucose orsaccharose solutions, Hank's solution, and other aqueous physiologicallybalanced salt solutions. The pH of the composition of the disclosure issuitably adjusted and buffered in order to be appropriate for use inhumans or animals, typically at a physiological or slightly basic pH(between about pH 8 to about pH 9, with a special preference for pH8.5). Suitable buffers include phosphate buffer (e.g. PBS), bicarbonatebuffer and/or Tris buffer. A typical composition is formulated in 1Msaccharose, 150 mM NaCl, 1 mM MgCl₂, 54 mg/l Tween 80, 10 mM Tris pH8.5. Another typical composition is formulated in 10 mg/ml mannitol, 1mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl.

The pharmaceutical formulation can also include any type ofpharmaceutically acceptable excipients, additives or vehicles. Forexample, but not by way of limitation, diluents or fillers, such asdextrates, dicalcium phosphate, calcium sulfate, lactose, cellulose,kaolin, mannitol, sodium chloride, dry starch, sorbitol, sucrose,inositol, powdered sugar, bentonite, microcrystalline cellulose, orhydroxypropylmethylcellulose may be added to the composition comprisingan extract or one or more compounds in an extract disclosed herein toincrease the bulk of the composition. Also, binders, such as but notlimited to, starch, gelatin, sucrose, glucose, dextrose, molasses,lactose, acacia gum, sodium alginate, extract of Irish moss, panwar gum,ghatti gum, mucilage of isapgol husks, carboxymethylcellulose,methylcellulose, polyvinylpyrrolidone, Veegum and starch arabogalactan,polyethylene glycol, ethylcellulose, and waxes, may be added to theformulation to increase its cohesive qualities. Additionally,lubricants, such as but not limited to, talc, magnesium stearate,calcium stearate, stearic acid, hydrogenated vegetable oils,polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride,leucine, carbowax, sodium lauryl sulfate, and magnesium lauryl sulfatemay be added to the formulation. Also, glidants, such as but not limitedto, colloidal silicon dioxide or talc may be added to improve the flowcharacteristics of a powdered formulation. Finally, disintegrants, suchas but not limited to, starches, clays, celluloses, algins, gums,crosslinked polymers (e.g., croscarmelose, crospovidone, and sodiumstarch glycolate), Veegum, methylcellulose, agar, bentonite, celluloseand wood products, natural sponge, cation-exchange resins, alginic acid,guar gum, citrus pulp, carboxymethylcellulose, or sodium lauryl sulfatewith starch may also be added to facilitate disintegration of theformulation in the intestine.

In certain embodiments, the formulation contains a directly compressiblecomposition comprising an extract or one or more compounds in an extractdisclosed herein but no excipients, additives or vehicles other than anenteric coating; however, the formulation may contain a lubricant, suchas but not limited to, magnesium stearate. Preferably, the directlycompressed formulation is formulated as a tablet of pharmaceuticallyacceptable hardness (greater than 6 kp, preferably 8-14 kp, and morepreferably 10-13 kp).

Polymers which are useful for the preparation of enteric coatingsinclude, but are not limited to, shellac, starch and amylose acetatephthalates, styrine-maleic acid copolymers, cellulose acetate succinate,cellulose acetate phthalate (CAP), polyvinylacetate phthalate (PVAP),hydroxypropylmethylcellulose phthalate (grades HP-50 and HP-55),ethylcellulose, fats, butyl stearate, and methacrylic acid-methacrylicacid ester copolymers with acid ionizable groups (“EUDRAGIT™”), such as“EUDRAGIT™ L 30D”, “EUDRAGIT™ RL 30D”, “EUDRAGIT™ RS 30D”, “EUDRAGIT™ L100-55”, and “EUDRAGIT™ L 30D-55”.

Application of the enteric coating to composition can be accomplished byany method known in the art for applying enteric coatings. For example,but not by way of limitation, the enteric polymers can be applied usingorganic solvent based solutions containing from 5 to 10% w/w polymer forspray applications and up to 30% w/w polymer for pan coatings. Solventsthat are commonly in use include, but are not limited to, acetone,acetone/ethyl acetate mixtures, methylene chloride/methanol mixtures,and tertiary mixtures containing these solvents. Some enteric polymers,such as methacrylic acid-methacrylic acid ester copolymers can beapplied using water as a dispersant.

Furthermore, plasticizers can be added to the enteric coating to preventcracking of the coating film. Suitable plasticizers include the lowmolecular weight phthalate esters, such as diethyl phthalate, acetylatedmonoglycerides, triethyl citrate, polyethyl glycoltributyl citrate andtriacetin. Generally, plasticizers are added at a concentration of 10%by weight of enteric coating polymer weight. Other additives such asemulsifiers, for example detergents and simethicone, and powders, forexample talc, may be added to the coating to improve the strength andsmoothness of the coating. Additionally, pigments may be added to thecoating to add color to the pharmaceutical formulation.

In certain embodiments, the composition comprising an extract or one ormore compounds in an extract disclosed herein is formulated with acompound or compounds which neutralize stomach acid. Alternatively, thepharmaceutical composition containing an extract or one or morecompounds in an extract disclosed herein is administered eitherconcurrent with or subsequent to administration of a pharmaceuticalcomposition which neutralize stomach acid. Compounds, such as antacids,which are useful for neutralizing stomach acid include, but are notlimited to, aluminum carbonate, aluminum hydroxide, bismuth subnitrate,bismuth subsalicylate, calcium carbonate, dihydroxyaluminum sodiumcarbonate, magaldrate, magnesium carbonate, magnesium hydroxide,magnesium oxide, and mixtures thereof.

In certain embodiments, composition comprising an extract or one or morecompounds in an extract disclosed herein is administered with asubstance that inactivates or inhibits the action of stomach enzymes,such as pepsin. Alternatively, the pharmaceutical composition containingthe proanthocyanidin polymer composition is administered eitherconcurrent with or subsequent to administration of a pharmaceuticalcomposition active to inactivate or inhibit the action of stomachenzymes. For example, but not by way of limitation, protease inhibitors,such as aprotin, can be used to inactivate stomach enzymes.

In certain embodiments, the composition comprising an extract or one ormore compounds in an extract disclosed herein is formulated with acompound or compounds which inhibit the secretion of stomach acid.Alternatively, the pharmaceutical composition is administered eitherconcurrent with or subsequent to administration of a pharmaceuticalcomposition active to inhibit the secretion of stomach acid. Compoundswhich are useful for inhibiting the secretion of stomach acid include,but are not limited to, ranitidine, nizatidine, famotidine, cimetidine,and misoprostol.

Cosmetic Formulations and Personal Care Products

In certain embodiments, the disclosure relates to a cosmetic formulationcomprising an extract or one or more compounds in an extract disclosedherein and cosmetically acceptable excipient or additive. In certainembodiments, the disclosure relates to a solid or liquid soap or lotioncomprising an extract or one or more compounds in an extract disclosedherein and a fatty acid.

In certain embodiments, additives can be selected from the groupconsisting of oily bodies, surfactants, emulsifiers, fats, waxes,pearlescent waxes, bodying agents, thickeners, superfatting agents,stabilizers, polymers, silicone compounds, lecithins, phospholipids,biogenic active ingredients, deodorants, antimicrobial agents,antiperspirants, film formers, antidandruff agents, swelling agents,insect repellents, hydrotropes, solubilizers, preservatives, perfumeoils and dyes.

In certain embodiments, additives are selected from the group consistingof surfactants, emulsifiers, fats, waxes, stabilizers, deodorants,antiperspirants, antidandruff agents and perfume oils.

As used herein, cosmetic preparations can mean care agents. Care agentsare understood as meaning care agents for skin and hair. These careagents include, inter alia, cleansing and restorative action for skinand hair.

In certain embodiments, preparations may be cosmetic and/ordermopharmaceutical preparations, e. g. hair shampoos, hair lotions,foam baths, shower baths, creams, gels, lotions, alcoholic andaqueous/alcoholic solutions, emulsions, wax/fat compositions, stickpreparations, powders or ointments.

Surfactants (or Surface-active substances) that may be present areanionic, non-ionic, cationic and/or amphoteric surfactants, the contentof which in the compositions is usually about 1 to 70% by weight,preferably 5 to 50% by weight and in particular 10 to 30% by weight.Typical examples of anionic surfactants are soaps,alkylbenzenesulfonates, alkanesulfonates, olefin sulfonates, alkyl ethersulfonates, glycerol ether sulfonates, a-methyl ester sulfonates, sulfofatty acids, alkyl sulphates, fatty alcohol ether sulphates, glycerolether sulphates, fatty acid ether sulphates, hydroxy mixed ethersulphates, monoglyceride (ether) sulphates, fatty acid amide (ether)sulphates, mono- and dialkyl sulfosuccinates, mono- and dialkylsulfosuccinamates, sulfotriglycerides, amide soaps, ether carboxylicacids and salts thereof, fatty acid isethionates, fatty acidsarcosinates, fatty acid taurides, N-acylamino acids, e.g. acyllactylates, acyl tartrates, acyl glutamates and acyl aspartates, alkyloligoglucoside sulphates, protein fatty acid condensates (in particularwheat-based vegetable products) and alkyl (ether) phosphates. If theanionic surfactants contain polyglycol ether chains, these may have aconventional homologous distribution, but preferably have a narrowedhomologous distribution. Typical examples of non-ionic surfactants arefatty alcohol polyglycol ethers, alkylphenol polyglycol ethers, fattyacid polyglycol esters, fatty acid amide polyglycol ethers, fatty aminepolyglycol ethers, alkoxylated triglycerides, mixed ethers or mixedformals, optionally partially oxidized alk(en)yl oligoglycosides orglucoronic acid derivatives, fatty acid N-alkylglucamides, proteinhydrolysates (in particular wheat-based vegetable products), polyolfatty acid esters, sugar esters, sorbitan esters, polysorbates and amineoxides. If the non-ionic surfactants contain polyglycol ether chains,these may have a conventional homologous distribution, but preferablyhave a narrowed homologous distribution. Typical examples of cationicsurfactants are quaternary ammonium compounds, e.g.dimethyldistearyl-ammonium chloride, and ester quats, in particularquaternized fatty acid trialkanolamine ester salts. Typical examples ofamphoteric or zwitterionic surfactants are alkylbetaines,alkylamidobetaines, aminopropionates, aminoglycinates,imidazolinium-betaines and sulfobetaines. Said surfactants are knowncompounds. With regard to structure and preparation of these substances,reference may be made to relevant review works.

Typical examples of particularly suitable mild, i.e. particularlyskin-compatible surfactants are fatty alcohol polyglycol ethersulphates, monoglyceride sulphates, mono- and/or dialkylsulfosuccinates, fatty acid isethionates, fatty acid sarcosinates, fattyacid taurides, fatty acid glutamates, α-olefinsulfonates, ethercarboxylic acids, alkyl oligoglucosides, fatty acid glucamides,alkylamidobetaines, amphoacetals and/or protein fatty acid condensates,the latter preferably based on wheat proteins.

Suitable oily bodies are, for example, alcohols based on fatty alcoholshaving 6 to 18, preferably 8 to 10, carbon atoms, esters of linearC₆-C₂₂-fatty acids with linear or branched C₆-C₂₂-fatty alcohols oresters of branched C₆-C₁₃-carboxylic acids with linear or branchedC₆-C₂₂-fatty alcohols, for example myristyl myristate, myristylpalmitate, myristyl stearate, myristyl isostearate, myristyl oleate,myristyl behenate, myristyl erucate, cetyl myristate, cetyl palmitate,cetyl stearate, cetyl isostearate, cetyl oleate, cetyl behenate, cetylerucate, stearyl myristate, stearyl palmitate, stearyl stearate, stearylisostearate, stearyl oleate, stearyl behenate, stearyl erucate,isostearyl myristate, isostearyl palmitate, isostearyl stearate,isostearyl isostearate, isostearyl oleate, isostearyl behenate,isostearyl oleate, oleyl myristate, oleyl palmitate, oleyl stearate,oleyl isostearate, oleyl oleate, oleyl behenate, oleyl erucate, behenylmyristate, behenyl palmitate, behenyl stearate, behenyl isostearate,behenyl oleate, behenyl behenate, behenyl erucate, erucyl myristate,erucyl palmitate, erucyl stearate, erucyl isostearate, erucyl oleate,erucyl behenate and erucyl erucate. Also suitable are esters of linearC₆-C₂₂-fatty acids with branched alcohols, in particular 2-ethylhexanol,esters of C₁₈-C₃₈-alkylhydroxycarboxylic acids with linear or branchedC₆-C₂₂-fatty alcohols, in particular dioctyl malates, esters of linearand/or branched fatty acids with polyhydric alcohols (for examplepropylene glycol, dimerdiol or trimertriol) and/or Guerbet alcohols,triglycerides based on C₆-C₁₀-fatty acids, liquid mono-/di-/triglyceridemixtures based on C₆-C₁₈-fatty acids, esters of C₆-C₂₂-fatty alcoholsand/or Guerbet alcohols with aromatic carboxylic acids, in particularbenzoic acid, esters of C₂-C₁₂-dicarboxylic acids with linear orbranched alcohols having 1 to 22 carbon atoms or polyols having 2 to 10carbon atoms and 2 to 6 hydroxyl groups, vegetable oils, branchedprimary alcohols, substituted cyclohexanes, linear and branchedC₆-C₂₂-fatty alcohol carbonates, for example dicaprylyl carbonates(Cetiol® CC), Guerbet carbonates based on fatty alcohols having 6 to 18,preferably 8 to 10, carbon atoms, esters of benzoic acid with linearand/or branched C₆-C₂₂-alcohols (e.g. Finsolv® TN), linear or branched,symmetrical or unsymmetrical dialkyl ethers having 6 to 22 carbon atomsper alkyl group, for example dicaprylyl ether (Cetiol® OE), ring-openingproducts of epoxidized fatty acid esters with polyols, silicone oils(cyclomethicones, silicon methicone types, inter alia) and/or aliphaticor naphthenic hydrocarbons, for example squalane, squalene ordialkylcyclohexanes.

Suitable emulsifiers are, for example, nonionogenic surfactants from atleast one of the following groups:

addition products of from 2 to 30 mol of ethylene oxide and/or 0 to 5mol of propylene oxide onto linear fatty alcohols having 8 to 22 carbonatoms, onto fatty acids having 12 to 22 carbon atoms, onto alkylphenolshaving 8 to 15 carbon atoms in the alkyl group, and onto alkylamineshaving 8 to 22 carbon atoms in the alkyl radical;

alkyl and/or alkenyl oligoglycosides having 8 to 22 carbon atoms in thealk(en)yl radical and the ethoxylated analogs thereof;

addition products of from 1 to 15 mol of ethylene oxide onto castor oiland/or hydrogenated castor oil;

addition products of from 15 to 60 mol of ethylene oxide onto castor oiland/or hydrogenated castor oil;

partial esters of glycerol and/or sorbitan with unsaturated, linear orsaturated, branched fatty acids having 12 to 22 carbon atoms and/orhydroxycarboxylic acids having 3 to 18 carbon atoms, and the adductsthereof with 1 to 30 mol of ethylene oxide;

partial esters of polyglycerol (average degree of self-condensation 2 to8), polyethylene glycol (molecular weight 400 to 5 000),trimethylolpropane, pentaerythritol, sugar alcohols (e.g. sorbitol),alkyl glucosides (e.g. methyl glucoside, butyl glucoside, laurylglucoside), and polyglucosides (e.g. cellulose) with saturated and/orunsaturated, linear or branched fatty acids having 12 to 22 carbon atomsand/or hydroxycarboxylic acids having 3 to 18 carbon atoms, and theadducts thereof with 1 to 30 mol of ethylene oxide;

mixed esters of pentaerythritol, fatty acids, citric acid and fattyalcohols and/or mixed esters of fatty acids having 6 to 22 carbon atoms,methylglucose and polyols, preferably glycerol or polyglycerol,

mono-, di- and trialkyl phosphates, and mono-, di- and/or tri-PEG alkylphosphates and salts thereof;

wool wax alcohols;

polysiloxane-polyalkyl-polyether copolymers and correspondingderivatives;

block copolymers, e.g. polyethylene glycol-30 dipolyhydroxystearates;

polymer emulsifiers, e.g. Pemulen® grades (TR-1, TR-2) from Goodrich;

polyalkylene glycols and glycerol carbonate.

The addition products of ethylene oxide and/or of propylene oxide ontofatty alcohols, fatty acids, alkylphenols or onto castor oil are known,commercially available products. These are homologous mixtures whoseaverage degree of alkoxylation corresponds to the ratio of the amountsof ethylene oxide and/or propylene oxide and substrate with which theaddition reaction is carried out. C12/18-fatty acid mono- and diestersof addition products of ethylene oxide onto glycerol are known asrefatting agents for cosmetic preparations.

Alkyl and/or alkenyl oligoglycosides can be prepared by reacting glucoseor oligosaccharides with primary alcohols having 8 to 18 carbon atoms.With regard to the glycoside radical, both monoglycosides, in which acyclic sugar radical is glycosidically bonded to the fatty alcohol, andalso oligomeric glycosides having a degree of oligomerization of up to,preferably, about 8, are suitable. The degree of oligomerization here isa statistical average value that is based on a homologous distributioncustomary for such technical-grade products.

Typical examples of suitable partial glycerides are hydroxy stearic acidmonoglyceride, hydroxy stearic acid diglyceride, isostearic acidmonoglyceride, isostearic acid diglyceride, oleic acid monoglyceride,oleic acid diglyceride, ricinoleic acid monoglyceride, ricinoleic aciddiglyceride, linoleic acid monoglyceride, linoleic acid diglyceride,linoleic acid monoglyceride, linoleic acid diglyceride, erucic acidmonoglyceride, erucic acid diglyceride, tartaric acid monoglyceride,tartaric acid diglyceride, citric acid monoglyceride, citric aciddiglyceride, malic acid mono-glyceride, malic acid diglyceride, and thetechnical-grade mixtures thereof which may also comprise small amountsof triglyceride as a minor product of the preparation process. Likewisesuitable are addition products of 1 to 30 mol, preferably 5 to 10 mol,of ethylene oxide onto said partial glycerides.

Suitable sorbitan esters are sorbitan monoisostearate, sorbitansesquiisostearate, sorbitan diisostearate, sorbitan triisostearate,sorbitan monooleate, sorbitan sesquioleate, sorbitan dioleate, sorbitantrioleate, sorbitan monoerucate, sorbitan sesquierucate, sorbitandierucate, sorbitan trierucate, sorbitan monoricinoleate, sorbitansesquiricinoleate, sorbitan diricinoleate, sorbitan triricinoleate,sorbitan monohydroxystearate, sorbitan sesquihydroxystearate, sorbitandihydroxystearate, sorbitan trihydroxystearate, sorbitan monotartrate,sorbitan sesquitartrate, sorbitan ditartrate, sorbitan tritartrate,sorbitan monocitrate, sorbitan sesquicitrate, sorbitan dicitrate,sorbitan tricitrate, sorbitan monomaleate, sorbitan sesquimaleate,sorbitan dimaleate, sorbitan trimaleate, and technical-grade mixturesthereof. Likewise suitable are addition products of 1 to 30 mol,preferably 5 to 10 mol, of ethylene oxide onto said sorbitan esters.

Typical examples of suitable polyglycerol esters are polyglyceryl-2dipolyhydroxystearate (Dehymuls® PGPH), polyglycerol-3 diisostearate(Lameform® TGI), polyglyceryl-4 isostearate (Isolan® GI 34),polyglyceryl-3 oleate, diisostearoyl polyglyceryl-3 diisostearate(Isolan® PDI), polyglyceryl-3 methylglucose distearate (Tego Care® 450),polyglyceryl-3 beeswax (Cera Bellina®), polyglyceryl-4 caprate(Polyglycerol Caprate T2010/90), polyglyceryl-3 cetyl ether (Chimexane®NL), polyglyceryl-3 distearate (Cremophor® GS 32) and polyglycerylpolyricinoleate (Admul® WOL 1403), polyglyceryl dimerate isostearate,and mixtures thereof. Examples of further suitable polyol esters are themono-, di- and triesters, optionally reacted with 1 to 30 mol ofethylene oxide, of trimethylolpropane or pentaerythritol with lauricacid, coconut fatty acid, tallow fatty acid, palmitic acid, stearicacid, oleic acid, behenic acid and the like.

Furthermore, zwitterionic surfactants can be used as emulsifiers. Theterm “zwitterionic surfactants” refers to those surface-active compoundsthat carry at least one quaternary ammonium group and at least onecarboxylate and one sulfonate group in the molecule. Particularlysuitable zwitterionic surfactants are the betaines, such asN-alkyl-N,N-dimethylammonium glycinates, for examplecocoalkyldimethylammonium glycinate,N-acylaminopropyl-N,N-dimethylammonium glycinates, for examplecocoacyl-amino-propyldimethylammonium glycinate, and2-alkyl-3-carboxymethyl-3-hydroxyethylimidazolines having in each case 8to 18 carbon atoms in the alkyl or acyl group, andcocoacylamino-ethylhydroxyethyl-carboxymethyl glycinate. Particularpreference is given to the fatty acid amide derivative known under theCTFA name Cocamidopropyl Betaine. Likewise suitable emulsifiers areampholytic surfactants. The term “ampholytic surfactants” means thosesurface-active compounds that, apart from a C_(8/18)-alkyl or -acylgroup in the molecule, contain at least one free amino group and atleast one —CO₂H or —SO₃H group and are capable of forming internalsalts. Examples of suitable ampholytic surfactants are N-alkylglycines,N-alkylpropionic acids, N-alkylaminobutyric acids,N-alkyliminodipropionic acids,N-hydroxyethyl-N-alkylamidopropylglycines, N-alkyl-taurines,N-alkylsarcosines, 2-alkylaminopropionic acids and alkylaminoaceticacids having in each case about 8 to 18 carbon atoms in the alkyl group.Particularly preferred ampholytic surfactants are N-cocoalkylaminopropionate, cocoacyl-aminoethyl aminopropionate andC_(12/18)-acylsarcosine. Finally, cationic surfactants are also suitableemulsifiers, those of the ester quat type, preferably methyl-quaternizeddifatty acid triethanolamine ester salts, being particularly preferred.

Fats and waxes that can be used are described in the following text.Typical examples of fats are glycerides, i.e. solid or liquid vegetableor animal products which consist essentially of mixed glycerol esters ofhigher fatty acids, suitable waxes are inter alia natural waxes, forexample candelilla wax, carnauba wax, japan wax, esparto grass wax, corkwax, guaruma wax, rice germ oil wax, sugarcane wax, ouricury wax, montanwax, beeswax, shellac wax, spermaceti, lanolin (wool wax), uropygialgrease, ceresin, ozokerite (earth wax), petrolatum, paraffin waxes,microcrystalline waxes; chemically modified waxes (hard waxes), forexample montan ester waxes, sasol waxes, hydrogenated jojoba waxes, andsynthetic waxes, for example polyalkylene waxes and polyethylene glycolwaxes. In addition to the fats, suitable additives are also fat-likesubstances, such as lecithins and phospholipids.

The term “lecithins” is understood by the person skilled in the art asmeaning those glycerophospholipids which form from fatty acids,glycerol, phosphoric acid and choline by esterification. Lecithins arethus frequently also known as phosphatidylcholines (PC). Examples ofnatural lecithins which may be mentioned are the cephalins, which arealso referred to as phosphatidic acids and represent derivatives of1,2-diacyl-sn-glycerol-3-phosphoric acids. By contrast, phospholipidsare usually understood as meaning mono- and, preferably, diesters ofphosphoric acid with glycerol (glycerophosphates), which are generallyconsidered to be fats. In addition, sphingosines and sphingolipids arealso suitable.

Examples of suitable pearlescent waxes are: alkylene glycol esters,specifically ethylene glycol distearate; fatty acid alkanolamides,specifically coconut fatty acid diethanolamide; partial glycerides,specifically stearic acid monoglyceride; esters of polybasic, optionallyhydroxy-substituted carboxylic acids with fatty alcohols having 6 to 22carbon atoms, specifically long-chain esters of tartaric acid; fattysubstances, for example fatty alcohols, fatty ketones, fatty aldehydes,fatty ethers and fatty carbonates, which have a total of at least 24carbon atoms, specifically laurone and distearyl ether; fatty acids,such as stearic acid, hydroxystearic acid or behenic acid, ring-openingproducts of olefin epoxides having 12 to 22 carbon atoms with fattyalcohols having 12 to 22 carbon atoms and/or polyols having 2 to 15carbon atoms and 2 to 10 hydroxyl groups, and mixtures thereof.

Bodying agents and thickeners that can be used are described in thefollowing text. Suitable bodying agents are primarily fatty alcohols orhydroxy fatty alcohols having 12 to 22, and preferably 16 to 18, carbonatoms, and also partial glycerides, fatty acids or hydroxy fatty acids.Preference is given to a combination of these substances with alkyloligoglucosides and/or fatty acid N-methylglucamides of identical chainlength and/or polyglycerol poly-12-hydroxystearates. Suitable thickenersare, for example, aerosil grades (hydrophilic silicas), polysaccharides,in particular xanthan gum, guar guar, agar agar, alginates and Tyloses,carboxymethylcellulose and hydroxyethylcellulose, and also relativelyhigh molecular weight polyethylene glycol mono- and diesters of fattyacids, polyacrylates (e.g. Carbopols® and Pemulen grades from Goodrich;Synthalens® from Sigma; Keltrol grades from Kelco; Sepigel grades fromSeppic; Salcare grades from Allied Colloids), polyacrylamides, polymers,polyvinyl alcohol and polyvinylpyrrolidone, surfactants, for exampleethoxylated fatty acid glycerides, esters of fatty acids with polyolsfor example pentaerythritol or trimethylolpropane, fatty alcoholethoxylates having a narrowed homolog distribution or alkyloligoglucosides, and electrolytes such as sodium chloride and ammoniumchloride.

Superfatting agents which can be used are for example lanolin andlecithin, and polyethoxylated or acylated lanolin and lecithinderivatives, polyol fatty acid esters, monoglycerides and fatty acidalkanolamides, the latter also serving as foam stabilizers.

Stabilizers which can be used are metal salts of fatty acids, forexample magnesium, aluminium and/or zinc stearate or ricinoleate.

Polymers that can be used are described in the following text. Suitablecationic polymers are, for example, cationic cellulose derivatives, forexample a quaternized hydroxyethylcellulose obtainable under the namePolymer JR 400® from Amerchol, cationic starch, copolymers ofdiallylammonium salts and acryl amides, quaternizedvinylpyrrolidone-vinylimidazole polymers, for example Luviquat® (BASF),condensation products of polyglycols and amines, quaternized collagenpolypeptides, for example lauryldimonium hydroxypropyl hydrolysedcollagen (Lamequat® L/Grunau), quaternized wheat polypeptides,polyethyleneimine, cationic silicone polymers, for exampleamodimethicones, copolymers of adipic acid anddimethylaminohydroxypropyldiethylenetriamine (Cartaretins®/Sandoz),copolymers of acrylic acid with dimethyl diallylammonium chloride(Merquat® 550/Chemviron), polyaminopolyamides and cross linkedwater-soluble polymers thereof, cationic chitin derivatives, for examplequaternized chitosan, optionally in microcrystalline dispersion,condensation products from dihaloalkyls, for example dibromobutane withbisdialkylamines, for example bis-dimethylamino-1,3-propane, cationicguar gum, for example Jaguar® CBS, Jaguar® C-17, Jaguar® C-16 fromCelanese, quaternized ammonium salt polymers, for example Mirapol® A-15,Mirapol® AD-1, Mirapol® AZ-1 from Miranol.

Suitable anionic, zwitterionic, amphoteric and nonionic polymers are,for example, vinyl acetate-crotonic acid copolymers,vinylpyrrolidone-vinyl acrylate copolymers, vinyl acetate-butylmaleate-isobornyl acrylate copolymers, methyl vinyl ether-maleicanhydride copolymers and esters thereof, uncrosslinked polyacrylic acidsand polyacrylic acids crosslinked with polyols,acrylamidopropyltrimethylammonium chloride-acrylate copolymers,octylacrylamide-methyl methacrylate-tert-butylamino-ethylmethacrylate-2-hydroxypropyl methacrylate copolymers,polyvinylpyrrolidone, vinylpyrrolidone-vinyl acetate copolymers,vinylpyrrolidone-dimethylaminoethyl methacrylate-vinylcaprolactamterpolymers, and optionally derivatized cellulose ethers and silicones.

Suitable silicone compounds are, for example, dimethylpolysiloxanes,methylphenylpolysiloxanes, cyclic silicones, and amino-, fatty-acid-,alcohol-, polyether-, epoxy-, fluorine-, glycoside- and/oralkyl-modified silicone compounds, which can either be liquid or inresin form at room temperature. Also suitable are simethicones, whichare mixtures of dimethicones having an average chain length of from 200to 300 dimethylsiloxane units and hydrogenated silicates.

Deodorants and antimicrobial agents that can be used are described inthe following text. Cosmetic deodorants counteract, mask or remove bodyodors. Body odors arise as a result of the effect of skin bacteria onapocrine perspiration, with the formation of degradation products whichhave an unpleasant odor. Accordingly, deodorants comprise activeingredients which act as antimicrobial agents, enzyme inhibitors, odorabsorbers or odor masking agents. Suitable antimicrobial agents are, inprinciple, all substances effective against gram-positive bacteria, forexample 4-hydroxybenzoic acid and its salts and esters,N-(4-chlorophenyl)-N′-(3,4-dichloro-phenyl)urea,2,4,4′-trichloro-2′-hydroxydiphenyl ether (triclosan),4-chloro-3,5-dimethylphenol, 2,2′-methylenebis(6-bromo-4-chlorophenol),3-methyl-4-(1-methyl-ethyl)phenol, 2-benzyl-4-chlorophenol,3-(4-chlorophenoxy)-1,2-propanediol, 3-iodo-2-propynyl butylcarbamate,chlorohexidine, 3,4,4′-trichlorocarbanilide (TTC), antibacterialfragrances, thymol, thyme oil, eugenol, oil of cloves, menthol, mintoil, famesol, phenoxyethanol, glycerol monocaprate, glycerolmonocaprylate, glycerol monolaurate (GML), diglycerol monocaprate (DMC),salicylic acid N-alkylamides, for example n-octylsalicylamide orn-decylsalicylamide.

Suitable enzyme inhibitors are, for example, esterase inhibitors. Theseare preferably trialkyl citrates, such as trimethyl citrate, tripropylcitrate, triisopropyl citrate, tributyl citrate and, in particular,triethyl citrate (Hydagen® CAT). The substances inhibit enzyme activity,thereby reducing the formation of odor. Other substances which aresuitable esterase inhibitors are sterol sulfates or phosphates, forexample lanosterol, cholesterol, campesterol, stigmasterol andsitosterol sulfate or phosphate, dicarboxylic acids and esters thereof,for example glutaric acid, monoethyl glutarate, diethyl glutarate,adipic acid, monoethyl adipate, diethyl adipate, malonic acid anddiethyl malonate, hydroxycarboxylic acids and esters thereof, forexample citric acid, malic acid, tartaric acid or diethyl tartrate, andzinc glycinate.

Suitable odor absorbers are substances which are able to absorb andlargely retain odor-forming compounds. They lower the partial pressureof the individual components, thus also reducing their rate ofdiffusion. It is important that in this process perfumes must remainunimpaired. Odor absorbers are not effective against bacteria. Theycomprise, for example, as main constituent, a complex zinc salt ofricinoleic acid or specific, largely odor-neutral fragrances which areknown to the person skilled in the art as “fixatives”, for exampleextracts of labdanum or styrax or certain abietic acid derivatives. Theodor masking agents are fragrances or perfume oils, which, in additionto their function as odor masking agents, give the deodorants theirrespective fragrance note. Perfume oils which may be mentioned are, forexample, mixtures of natural and synthetic fragrances. Naturalfragrances are extracts from flowers, stems and leaves, fruits, fruitpeels, roots, woods, herbs and grasses, needles and branches, and resinsand balsams. Also suitable are animal raw materials, for example civetand castoreum. Typical synthetic fragrance compounds are products of theester, ether, aldehyde, ketone, alcohol and hydro-carbon type. Fragrancecompounds of the ester type are, for example, benzyl acetate,p-tert-butylcyclohexyl acetate, linalyl acetate, phenylethyl acetate,linalyl benzoate, benzyl formate, allyl cyclohexylpropionate, styrallylpropionate and benzyl salicylate. The ethers include, for example,benzyl ethyl ether, and the aldehydes include, for example, the linearalkanals having 8 to 18 carbon atoms, citral, citronellal,citronellyloxyacetaldehyde, cyclamen aldehyde, hydroxycitronellal,lilial and bourgeonal, the ketones include, for example, the ionones andmethyl cedryl ketone, the alcohols include anethole, citronellol,eugenol, isoeugenol, geraniol, linalool, phenylethyl alcohol andterpineol, and the hydrocarbons include mainly the terpenes and balsams.Preference is, however, given to using mixtures of different fragranceswhich together produce a pleasing fragrance note. Ethereal oils ofrelatively low volatility, which are mostly used as aroma components,are also suitable as perfume oils, e.g. sage oil, camomile oil, oil ofcloves, melissa oil, mint oil, cinnamon leaf oil, linden flower oil,juniper berry oil, vetiver oil, olibanum oil, galbanum oil, labdanum oiland lavandin oil. Preference is given to using bergamot oil,dihydromyrcenol, lilial, lyral, citronellol, phenyl ethyl alcohol,a-hexyl cinnamal dehy de, geraniol, benzylacetone, cyclamen aldehyde,linalool, boisambrene forte, ambroxan, indole, hedione, sandelice, lemonoil, mandarin oil, orange oil, allyl amyl glycolate, cyclovertal,lavandin oil, clary sage oil, β-damascone, geranium oil bourbon,cyclohexyl salicylate, Vertofix coeur, iso-E-super, Fixolide NP, evemyl,iraldein gamma, phenylacetic acid, geranyl acetate, benzyl acetate, roseoxide, romilat, irotyl and floramat alone or in mixtures.

Antiperspirants reduce the formation of perspiration by influencing theactivity of the eccrine sweat glands, thus counteracting underarmwetness and body odor. Aqueous or anhydrous formulations ofantiperspirants typically comprise one or more of the followingingredients: astringent active ingredients, oil components, nonionicemulsifiers, coemulsifiers, bodying agents, auxiliaries, for examplethickeners or complexing agents, and/or nonaqueous solvents, for exampleethanol, propylene glycol and/or glycerol.

Suitable astringent antiperspirant active ingredients are primarilysalts of aluminum, zirconium or of zinc. Such suitable antihydroticactive ingredients are, for example, aluminum chloride, aluminumchlorohydrate, aluminum di chl orohy drate, aluminum sesquichlorohydrateand complex compounds thereof, e.g. with 1,2-propylene glycol, aluminumhydroxyallantoinate, aluminum chloride tartrate, aluminum zirconiumtrichlorohydrate, aluminum zirconium tetrachlorohydrate, aluminumzirconium pentachlorohydrate and complex compounds thereof, e.g. withamino acids, such as glycine. In addition, customary oil-soluble andwater-soluble auxiliaries may be present in antiperspirants inrelatively small amounts. Such oil-soluble auxiliaries may, for example,be anti-inflammatory, skin-protective or perfumed ethereal oils,synthetic skin-protective active ingredients and/or oil-soluble perfumeoils.

Customary water-soluble additives are, for example, preservatives,water-soluble fragrances, pH regulators, e.g. buffer mixtures,water-soluble thickeners, e.g. water-soluble natural or syntheticpolymers, for example xanthan gum, hydroxyethylcellulose,polyvinylpyrrolidone or high molecular weight polyethylene oxides.

Film formers that can be used are described in the following text.Customary film formers are, for example, chitosan, microcrystallinechitosan, quaternized chitosan, polyvinylpyrrolidone,vinylpyrrolidone-vinyl acetate copolymers, polymers of the acrylic acidseries, quaternary cellulose derivatives, collagen, hyaluronic acid andsalts thereof, and similar compounds.

Suitable antidandruff active ingredients are piroctone olamine(1-hydroxy-4-methyl-6-(2,4,4-trimethylpentyl)-2-(1H)-pyridinonemonoethanolamine salt), Baypival® (climbazole), Ketoconazole®,(4-acetyl-1-{ -442-(2,4-dichlorophenyl) r-2-(1H-imidazol-1-ylmethyl]-1,3-dioxylan-c-4-ylmethoxyphenyl}piperazine, ketoconazole, elubiol,selenium disulfide, colloidal sulfur, sulfur polyethylene glycolsorbitan monooleate, sulfur ricinol polyethoxylate, sulfur tardistillates, salicyclic acid (or in combination with hexachlorophene),undecylenic acid monoethanolamide sulfosuccinate Na salt, Lamepon® UD(protein undecylenic acid condensate), zinc pyrithione, aluminumpyrithione and magnesium pyrithione/dipyrithione magnesium sulfate.

The swelling agents for aqueous phases may be montmorillonites, claymineral substances, Pemulen, and alkyl-modified Carbopol grades(Goodrich).

Suitable insect repellents are N,N-diethyl-m-toluamide, 1,2-pentanediolor ethyl butylacetylaminopropionate.

To improve the flow behavior, hydrotropes, for example ethanol,isopropyl alcohol, or polyols, can be used. Polyols which are suitablehere preferably have 2 to 15 carbon atoms and at least two hydroxylgroups. The polyols can also contain further functional groups, inparticular amino groups, or be modified with nitrogen. Typical examplesare:

glycerol;

alkylene glycols, for example, ethylene glycol, diethylene glycol,propylene glycol, butylene glycol, hexylene glycol, and polyethyleneglycols with an average molecular weight of from 100 to 1 000 daltons;

technical-grade oligoglycerol mixtures with a degree ofself-condensation of from 1.5 to 10, for example, technical-gradediglycerol mixtures with a diglycerol content of from 40 to 50% byweight;

methylol compounds, such as trimethylolethane, trimethylolpropane,trimethylol-butane, pentaerythritol and dipentaerythritol;

lower alkyl glucosides, in particular those with 1 to 8 carbon atoms inthe alkyl radical, for example methyl and butyl glucoside;

sugar alcohols with 5 to 12 carbon atoms, for example sorbitol ormannitol,

sugars with 5 to 12 carbon atoms, for example glucose or sucrose;

amino sugars, for example glucamine;

dialcohol amines, such as diethanolamine or 2-amino-1,3-propanediol.

Suitable preservatives are, for example, phenoxyethanol, formaldehydesolution, parabenes, pentanediol or sorbic acid, and the other classesof substance listed in Annex 6, Part A and B of the Cosmetics Directive.

Perfume oils which may be used are preferably mixtures of natural andsynthetic fragrances. Natural fragrances are extracts from flowers(lily, lavender, rose, jasmine, neroli, ylang-ylang), stems and leaves(geranium, patchouli, petitgrain), fruits (aniseed, coriander, cumin,juniper), fruit peels (bergamot, lemon, orange), roots (mace, angelica,celery, cardamom, costus, iris, calmus), woods (pine wood, sandalwood,guaiac wood, cedarwood, rosewood), herbs and grasses (tarragon, lemongrass, sage, thyme), needles and branches (spruce, fir, pine,dwarf-pine), resins and balsams (galbanum, elemi, benzoin, myrrh,olibanum, opoponax). Also suitable are animal raw materials, for examplecivet and castoreum. Typical synthetic fragrance compounds are productsof the ester, ether, aldehyde, ketone, alcohol and hydrocarbon type.Fragrance compounds of the ester type are, for example, benzyl acetate,phenoxyethyl isobutyrate, p-tert-butylcyclohexyl acetate, linalylacetate, dimethylbenzylcarbinyl acetate, phenylethyl acetate, linalylbenzoate, benzyl formate, ethylmethylphenyl glycinate, allylcyclohexylpropionate, styrallyl propionate and benzyl salicylate. Theethers include, for example, benzyl ethyl ether, the aldehydes include,for example, the linear alkanals having 8 to 18 carbon atoms, citral,citronellal, citronellyloxyacetaldehyde, cyclamen aldehyde,hydroxycitronellal, lilial and bourgeonal, and the ketones include, forexample, the ionones, a-isomethylionone and methyl cedryl ketone, thealcohols include anethole, citronellol, eugenol, isoeugenol, geraniol,linalool, phenylethyl alcohol and terpineol, and the hydrocarbonsinclude predominantly the terpenes and balsams. Preference is, however,given to using mixtures of different fragrances which together produce apleasing fragrance note. Ethereal oils of relatively low volatility,which are mostly used as aroma components, are also suitable as perfumeoils, e.g. sage oil, camomile oil, oil of cloves, melissa oil, mint oil,cinnamon leaf oil, linden blossom oil, juniper berry oil, vetiver oil,olibanum oil, galbanum oil, labolanum oil and lavandin oil. Preferenceis given to using bergamot oil, dihydromyrcenol, lilial, lyral,citronellol, phenylethyl alcohol, a-hexylcinnamaldehyde, geraniol,benzyl-acetone, cyclamen aldehyde, linalool, boisambrene forte,ambroxan, indole, hedione, sandelice, lemon oil, mandarin oil, orangeoil, allyl amyl glycolate, cyclovertal, lavandin oil, clary sage oil,β-damascone, geranium oil bourbon, cyclohexyl salicylate, Vertofixcoeur, iso-E-super, Fixolide NP, evemyl, iraldein gamma, phenylaceticacid, geranyl acetate, benzyl acetate, rose oxide, romilat, irotyl andfloramat alone or in mixtures.

Medical Device Coatings, Wound Dressings, and Irrigation

In certain embodiments, the disclosure relates to a medical devicecomprising a coating comprising an extract or one or more compounds inan extract disclosed herein optionally in combination with anotherantibiotic. In certain embodiments, the medical device is an ear tube,eye lenses, contact lenses, coronary stent, metal screw, pin, plate,rod, catheter, artificial knee, cardioverter defibrillator, artificialhip, heart pacemaker, breast implant, spine screws, rods, and discs,intra-uterine devices

In certain embodiments, the disclosure relates to a wound dressingcomprising an extract or one or more compounds in an extract disclosedherein wherein the wound dress comprises an absorbent pad and optionallyan adhesive optionally in combination with another antibiotic agent. Incertain embodiments, the wound dressing is a foam or compressiondressing or a cover dressing such as wraps, gauze and tape.

In certain embodiments, the wound dressing comprises alginate orcollagen.

In certain embodiments, the wound dressing a hydrocolloid dressing,e.g., carboxy-methylcellulose and gelatin optionally in a polyurethanefoam or film, optionally comprising one or more agents selected from,pectin, polysaccharides, and an adhesive.

In certain embodiments, the wound dressing is a hydrogel. Hydrogels arepolymers that contain a high content, e.g., greater than 40, 50, 60, 70,80, 90, or 95%, of hydroxy and/or carboxyl containing monomers or saltsthereof, e.g., vinyl alcohol, acrylic acid, 2-hydroxyethylmethacrylatemonomers, which can be co-polymers to provide varying degrees ofhydration, e.g., copolymerization with ethylene glycol dimethacrylate.Due to the hydrophilic monomers, the hydrogels typically absorb water tocontain greater than 70, 80, 85, 90, 95% water by weight. Contemplatedhydrogel dressings include: amorphous hydrogel, which are a free-flowinggel that are typically distributed in tubes, foil packets and spraybottles; an impregnated hydrogel, which are typically saturated onto agauze pad, nonwoven sponge ropes and/or strips; or a sheet hydrogelwhich are gel held together by a fiber mesh.

A flow of wound rinse/irrigation solution is applied across an openwound surface to achieve wound hydration, to remove deeper debris, andto assist with the visual examination. In certain embodiments, thedisclosure relates to methods of irrigating using a solution comprisingan extract or one or more compound in an extract disclosed herein. Incertain embodiments, the disclosure relates to a wound rinse comprisingan extract or one or more compounds in an extract disclosed hereinoptionally in combination with normal saline, sterile water, detergent,surfactant, preservatives, or iodine.

In certain embodiments, the disclosure contemplate a kit comprising acontainer comprising an extract or one or more compounds in an extractdiscloses herein optionally comprising a second container comprising asolution, normal saline, sterile water, detergent, surfactant,preservatives, iodine, hydrogen peroxide, or sodium hypochlorite.

EXAMPLES Collection and Crude Extraction of Plant Materials

Fresh leaves of the European Chestnut (Castanea sativa Mill., Fagaceae)were collected from wild populations in the months of May-July(2012-2014) in the Rionero-Alto Bradano region of the BasilicataProvince in southern Italy following standard guidelines for collectionof wild specimens. Collections were made on private land with thepermission of the landowner. Voucher specimens (CQ-309) were depositedat the Herbarium Lucanum (HLUC) at the University della Basilicata inPotenza, Italy and the Emory University Herbarium (GEO) in Atlanta, Ga.,USA. The specimens were identified using the standard Italian Flora andidentification was confirmed at HLUC. Chestnut leaves were shade-dried,ground with a blender, and vacuum sealed with silica packets prior toshipment to the US (under USDA permit) for extraction and analysis. Uponarrival at the lab, leaves were further ground into a fine powder with aThomas Wiley Mill at a 2 mm mesh size (Thomas Scientific).

Extraction and Purification of QSI-containing Fractions

Crude methanol extracts (Extract 224) of the ground leaves were createdby maceration of the plant materials at room temperature using a ratioof 1 g dry leaves: 10 mL MeOH for two successive periods of 72 hours,with daily agitation. Filtered extracts were combined, concentrated atreduced pressure and a temperature<40° C. with rotary evaporators, andlyophilized before being re-suspended in water and partitioned insuccession with hexane, ethyl acetate and butanol. The resultingnon-aqueous partitions were dried over anhydrous Na₂SO₄, concentrated invacuo, and lyophilized before testing for activity.

The most active partition (ethyl acetate, extract 224C) was subjected tofurther fractionation using a CombiFlash® Rf+ (Teledyne ISCO) flashchromatography system using a RediSep Rf Gold silica column. Extract224C was bonded to Celite 545 (Acros Organics) at a 1:4 ratio anddry-loaded using a RediSep dry load cartridge. The mobile phaseconsisted of (A) hexane, (B) EtOAc, and (C) MeOH. The linear gradientbegins with 100% A for 6.3 column volumes (CV), then 50:50 A:B at 25.3CV, to 100% B at 63.3 CV, which is held till 69.6 CV, then to 70:30 B:Cat 88.6 CV which is held till 94.9 CV. The chromatography was monitoredat 254 and 280 nm, as well as via ELSD. The resulting fractions werecombined into 5 fractions. Following further bioassay testing, it wasdetermined that the fraction which eluted from 30-40 CV (224C-F2) wasmost active. The full extract fractionation scheme is presented in FIG.2.

Characterization by HPLC and LC-FTMS

An analytical HPLC-method was developed for the purposes ofcharacterization of 224 and fractions. The analysis was performed on anAgilent 1260 Infinity system running OpenLab CDS ChemStation (AgilentTechnologies, Santa Clara, Calif., USA) with an Agilent ZORBAX EclipseXDB-C₁₈ (250 mm×4.6 mm, 5 μm) column with compatible guard column at acolumn temperature of 40° C. Mobile phase reagents were HPLC-grade andpurchased from Fisher Scientific, except for the Type 1 water, which wasobtained from an EMD Millipore MILLI-Q water system (Billerica, Mass.).Mobile phase consisted of a linear gradient elution 0.1% formic acid inacetonitrile (A) and 0.1% formic acid in water (B) at a flow rate of 1mL/min. Initial conditions were 98:2 (A:B) changing to 70:30 (A:B) at 50min, to 2:98 (A:B) at 70 min and held until 85min., Samples wereprepared in DMSO and 10 μL injections were made. Chromatograms weremonitored at 254 nm and 314 nm.

Liquid chromatography-Fourier transform mass spectrometry (LC-FTMS) wasperformed on 224C-F2 using a Shimadzu SIL-ACHT and Dionex 3600SD HPLCpump with a modification of the previous chromatographic conditions. A20 μL injection at ambient temperature with 0.1% formic acid in OptimaLC/MS acetonitrile (Fisher Scientific) (A) and 0.1% formic acid in water(B) at a flow rate of 1 mL/min. Initial conditions were 98:2 (A:B)changing to 64:36 (A:B) at 12 min, to 52:48 (A:B) at 86 min, 2:98 (A:B)at 102.6 min and held until 117.6 min before returning to initialconditions to equilibrate the column. The data was acquired in MS¹ modescanning from a m/z of 150-1500 on a Thermo Scientific LTQ-FT Ultra MSin negative ESI mode and processed with Thermo Scientific Xcalibur 2.2SP1.48 software (San Jose, Calif.). The capillary temperature was 275.0°C., sheath gas of 60, source voltage and current 5.0 kV and 100.0 μA,and the capillary voltage −49.0 V.

Putative compounds were determined for compounds present in thebioactive active region of 224C-F2′ s chromatogram (retention time of21-49 min). The Dictionary of Natural Products (CRC Press) and Scifinder(Chemical Abstracts Service) were searched in May 2015 using similarmethodology. The high resolution mass of the compound was determinedfrom the LC-FTMS data and the database searched for all compounds within±0.5 Da. The resulting compounds were limited to only those identifiedin the genus Castanea, for DNP several entries for the misspelling“Castaneae” were also included. The molecular formulas of the remainingcompounds were compared to empirical formulas derived from the MS dataand those that matched the experimental molecular mass with a delta ofless than 100 ppm were evaluated further. Only small molecules wereconsidered for further evaluation. Publications on the remaining smallmolecules were reviewed and the presence of the compound in the genuswas verified.

In addition to examining LC-FTMS data and fragmentation patterns asdescribed above, a number of natural products were specifically searchedfor in 224C-F2: chlorogenic acid, ellagic acid hyperoside, isoquercitrinand rutin. Standards of chlorogenic acid and ellagic acid (MPBiomedicals, Solon OH) and hyperoside (Chromadex, Irvine, CA) were runon the analytical HPLC method described above to determine retentiontimes. The others were examined by MS fragmentation patterns andpublished UV-Vis spectra. Standards were evaluated for purity viaHPLC-DAD.

Bacterial Strains, Plasmids, and Culture Media.

S. aureus cultures were grown in Tryptic Soy Broth (TSB) or Tryptic SoyAgar (TSA). Cation-adjusted Mueller-Hinton broth (CAMHB) was used forminimum inhibitory concentration (MIC) testing of S. aureus. Thebacterial strains and plasmids used in this study are described in Table1.

TABLE 1 Description of bacterial strains and plasmids used. DesignationSpecies Other Characteristics* AH408; SA502A Staphylococcus aureus agrgroup II AH430 Staphylococcus aureus SA502a + pDB59 cmR, yfp reporter,agr group II AH845 Staphylococcus aureus agr group I AH1263; LACStaphylococcus aureus CA-MRSA, PFT USA300, agr group I AH1677Staphylococcus aureus AH845 + pDB59 cmR, yfp reporter, agr group IAH1747 Staphylococcus aureus MW2 + pDB59 cmR, yfp reporter, agr groupIII AH1872 Staphylococcus aureus MN EV(407) + pDB59 cmR, yfp reporter,agr group IV AH2759 Staphylococcus aureus AH1263 agr P3:lux AH3052Staphylococcus aureus AH1263 Δspa F0392; HM-262 Streptococcus mitis HMP,oral cavity isolate FS1; NR-13441 Corynebacterium striatum Clinicalisolate from Italy, 2005- 2007 MGAS15252; NR- Streptococcus pyogenesserotype M59, Group A 33709 Streptococcus (GAS) HL005PA2; HM-493Propionibacterium acnes HMP, skin isolate MN EV(407) Staphylococcusaureus agr group IV MW-2 Staphylococcus aureus agr group III NIHLM001;HM- Staphylococcus epidermidis HMP, 2008 skin isolate from alar 896crease from healthy volunteer NRS-116; NR-45922 StaphylococcusGlycopeptide intermediate, 2002 haemolyticus surgical isolate NR5385;NR-46071 Staphylococcus aureus HA-MRSA, PFT USA500, MLST ST8, SCC mecIV,agr group I, sea+, seb+ SK46; HM-109 Corynebacterium HMP, skin isolateon arm of healthy amycolatum volunteer SK58; HM-114 Micrococcus luteusHMP, skin isolate on arm of healthy volunteer SK66; HM-120Staphylococcus warneri HMP, skin isolate on arm of healthy volunteerUAMS-1 Staphylococcus aureus MSSA, osteomyelitis isolate UAMS-929Staphylococcus aureus isogenic sarA mutant of UAMS-1

Escherichia coli cultures were grown in Luria-Bertani (LB) broth or onLB agar plates supplemented with 100 μg mL ⁻¹ ampicillin (Amp) asrequired for plasmid maintenance. S. aureus chromosomal markers orplasmids were selected for with 10 μg mL⁻¹ of chloramphenicol (Cam) orerythromycin (Erm). Staphylococcus warneri cultures were grown in TSB orBrain-Heart Infusion (BHI) agar. Micrococcus luteus cultures were grownin nutrient broth or agar. Streptococcus mitis, Streptococcus pyogenes,Corynebacterium amycolatum, Staphylococcus haemolyticus andStaphylococcus epidermidis cultures were grown in BHI broth or TSA with5% sheep blood. Corynebacterium striatum cultures were grown in TSB orTSA with 5% sheep blood. Propionibacterium acnes cultures were grown inReinforced Clostridial Medium (RCM) broth or TSA with 5% sheep bloodunder static, anaerobic conditions generated by GasPak EZ Systems.Unless otherwise stated, all broth cultures were grown at 37° C. withshaking at 250 rpm.

Minimum Inhibitory Concentration (MIC)

Extract 224 and fractions were examined for minimum inhibitoryconcentrations (MIC) against strains representing the four agr alleles(AH430, AH1677, AH1747, AH1872), biofilm test strain (UAMS-1) and aUSA500 strain (NRS385), which was used in 6-toxin quantificationexperiments. Clinical Laboratory Standards Institute (CL SI) M100-S23guidelines for microtiter broth dilution testing were followed. Controlsinclude the vehicle, and antibiotics: Kanamycin (Kan) and Amp (MPBiomedicals Inc). All concentrations were tested in triplicate andrepeated twice on different days. Briefly, overnight cultures in CAMHBwere standardized by OD to 5×10⁵ CFU/mL, and this was confirmed by platecounts. Two-fold serial dilutions were performed on a 96-well plate(Falcon 35-1172) to achieve a test range of 512-0.25 μg mL⁻¹ forextracts and 64-0.03125 μg mL⁻¹ for Amp and Kan. Plates were incubatedat 37° C. for 18 hrs. under static conditions. Plates were read at an OD600 nm in a Cytation 3 multimode plate reader (Biotek) at 0 and 18 hrs.post inoculation. The following formula, which takes into account theimpact of extract color and vehicle on the OD, was used as described inQuave et al., J Ethnopharmacol. 2008, 118(3):418-28:

$\; {{\% {\mspace{11mu} \;}{Inhibition}} = {\left\lbrack {1 - \left( \frac{{OD}_{{t\; 18} -}{OD}_{t\; 0}}{{OD}_{{{vc}\; 18} -}{OD}_{{vc}\; 0}} \right)} \right\rbrack \times 100}}$

with OD_(t18)=OD of the test well at 18 hrs., OD_(t0)=OD of the testwell at 0 hrs., OD_(vc18)=OD of the vehicle control well at 18 hrs, andOD_(vc0)=OD of the vehicle control well at 0 hrs. MIC₅₀ and MIC₉₀ valueswere assigned based on the concentration at which at least 50 or 90%inhibition of growth was observed as determined by OD, respectively.

Growth inhibition of the refined extract, 224C-F2, was also assessed forimpact on the normal skin microflora. In all cases, with the exceptionof P. acnes, the appropriate CLSI method for MIC determination by brothmicrodilution was employed. Briefly, MICs for Staphylococcus warneri, S.epidermidis, S. haemolyticus and Micrococcus luteus were determinedusing the above described M100-S23 CLSI method for S. aureus withvehicle and antibiotic controls. Amp and Kan (MP Biomedicals Inc) wereused in all staphylococcal tests; Amp, Erm (Sigma Aldrich) andclindamycin, Clin (MP Biomedicals) were used for M luteus controls. MICsfor Streptococcus pyogenes and S. mitis were determined using theM100-S23 CLSI method in CAMHB with 3% lysed horse blood (LHB), incubatedat 37° C. for 24 hrs under static conditions, with Amp and Erm asantibiotic controls. MICs for Corynebacterium striatum and C. amycolatumfollowed the M45-A2 CLSI method in CAMHB with 3% LHB, incubated at 35°C. for 24 hrs under static conditions, with Amp and Erm as antibioticcontrols. MICs for Propionibacterium acnes were based on a previousmethod using BHI supplemented with 1% dextrose, incubated at 37° C. for72 hrs under static, anaerobic conditions.

Quorum Quenching Assays with Reporter Strains

Extracts were tested for quorum quenching activity against all four agrtypes using agr P3-YFP reporter strains AH1677 (type I), AH430 (typeII), AH1747 (type III), and AH1872 (type IV), as well as agr P3-lux(type I) reporter strain AH2759. Overnight cultures of reporter strainsthat were grown in TSB supplemented with Cam were inoculated at adilution of 1:250 into fresh TSB containing Cam. 100 !IL aliquots wereadded to 96-well microtiter plates (Costar 3603) containing 100 μLaliquots of TSB containing Cam and 2-fold serial dilutions (0.1-200 μgmL⁻¹) of extracts 224, 224C, and 224C-F2. After mixing, the effectiveinoculum dilution was 1:500 and the final extract concentrations rangedfrom 0.05-100 μg mL⁻¹, with a final DMSO concentration of 1% (v/v) inall wells. Four dilution series were prepared for each reporter/extractcombination, and in addition 4 mock vehicle (DMSO) dilution series wereincluded for each reporter strain. Microtiter plates were incubated at37° C. with shaking (1000 rpm) in a Stuart SI505 incubator (BibbyScientific, Burlington, N.J.) with a humidified chamber. Fluorescence(top reading, 493 nm excitation, 535 nm emission, gain 60) and opticaldensity (OD) readings at 600 nm, or luminescence and OD600 readings inthe case of reporter AH2759, were recorded at 30 min increments using aTecan Systems (San Jose, CA) Infinite M200 plate reader.

Hemolytic Activity by Red Blood Cell Lysis Assay

The quorum quenching activity of extracts was assessed by measuring thehemolytic activity of culture supernatants on rabbit red blood celllysis. Overnight cultures of an Erm sensitive variant of USA300 strainLAC, AH1263 and an hla::Tn551 (AH1589) mutant of AH1263 were inoculated1:500 into 5 ml of TSB (in 17×150 mm culture tubes) containing extracts224, 224C, or 224CF2 at concentrations of 6.25, 12.5, 25, 50 and 100 μgmL⁻¹. In all tubes containing extract the mock vehicle (DMSO)concentration was held constant at 1% (v/v). Vehicle control tubescontaining 1% DMSO were similarly prepared for AH1263, AH1589 well asfor an Δagr::tetM (AH1292) mutant of AH1263. All tubes were incubated at37° C. with shaking (250 rpm), and growth was monitored by periodicallytransferring 100 μL of culture to a 96-well microtiter plate and readingOD600 in a Tecan Systems (San Jose, Calif.) Infinite M200 plate reader.Following 6 hrs of incubation, 600 μL of each culture was filtersterilized using cellulose acetate SpinX 0.22 μm filters (Corning).

To quantify hemolytic activity, the filter sterilized culturesupernatants were serially diluted in 2-fold steps (from 0.04-100%) inTSB, and 50 μL aliquots were dispensed in quadruplicate into 96-wellmicrotiter plates. Rabbit erythrocytes, prepared from defibrinated blood(Hemostat Laboratories, Dixon, Calif.) by washing 3 times with 1.1× PBSand resuspending in 1.1× PBS at 1% (v/v), were added to the microtiterplates at 50 μL per well (yielding a final erythrocyte concentration of0.5% (v/v)). The erythrocytes and culture supernatants were mixedthoroughly and incubated statically at room temperature for 2 hrs.Hemolysis was detected by the loss of turbidity as measured at OD63ousing a Tecan Systems (San Jose, Calif.) Infinite M200 plate reader.Relative hemolytic activities were obtained by using KaleidaGraph 4.1.3(Synergy Software, Reading, Pa., USA) to perform 4-parameter logisticfits of the turbidity data in order to determine the concentration ofsupernatant that resulted in 50% red blood cell lysis.

Western Blot for Alpha-hemolysin

An overnight culture of S. aureus AH3052 Δspa was inoculated into 5 mLof TSB at 1:500 and grown at 37° C. with shaking (250 rpm), in thepresence of either DMSO or one of the extracts (224, 224C or 224C-F2) atconcentrations of 6.25, 12.5, 25, 50 and 100 μg mL⁻¹. Following 8 hoursof incubation, 600 μL of each culture was filter sterilized using acellulose acetate SpinX 0.22 μm filter (Corning) and the filtersterilized media was stored at −20° C. The filtered media waselectrophoresed on 13% SDS-PAGE gels and transferred to nitrocellulosemembranes (Bio-Rad). Membranes were blocked overnight at 4° C. in TBST(20 mM Tris [pH 7.5], 150 mM NaCl, 0.1% Tween 20) with 5% nonfat drymilk then washed 3 times with TB ST. Hla was detected using a polyclonalrabbit anti-Hla antibody (Shlievert Lab, University of Iowa) at a 1:5000dilution and a goat anti-rabbit HRP secondary antibody (JacksonImmunoResearch Laboratories) at a 1:20000 dilution. Blots were incubatedat RT for 5 min with Supersignal West Pico Chemiluminescent Substrate(Thermo Scientific) then exposed to film for 30 min.

Resistance Passaging

To determine the ability of S. aureus to generate resistance to thequorum quenching effects of 224C-F2, cultures were exposed to sub-MICconcentrations (16 μg mL⁻¹) of extract for 15 hrs, the OD₆₀₀ taken, andcultures centrifuged. The cell-free supernatant was removed and frozenfor later HPLC quantification of 6-toxin. The cell pellets were thenreconstituted in TSB to an OD equivalent of 5×10⁵ CFU mL⁻¹ with extract(or vehicle control) added, and incubated while shaking as describedabove. This process was repeated for a total of 15 passaging days.

Biofilm Assay

Extract 224 and fractions were examined for impact on S. aureus biofilmformation using a human plasma protein-coated assay using strains UAMS-1(a PFGE USA200 osteomyelitis isolate, agr type III) and its isogenicsarA mutant, UAMS-929, which has a biofilm deficient phenotype andserves as a positive control. the natural product-based anti-biofilmcomposition “220D-F2” was also included which inhibits biofilm formationin both Staphylococcus aureus and Streptococcus pneumoniae, as apositive drug control. Following inoculation and addition of appropriatemedia (containing extract or vehicle alone), 96-well plates (Falcon35-1172) were incubated for 22 hrs at 37° C. The wells were gentlywashed with phosphate-buffered saline (PBS), fixed with ethanol, stainedwith crystal violet, rinsed in tap water, and the stain eluted intoethanol and transferred to a new plate prior to quantification of theeluate at an OD595 with a Cytation™ 3 multimode plate reader (Biotek).

Mice and S. aureus Skin Infection Model

C₅Bl/6 dams were purchased from Charles Rivers (Wilmington, Mass.). Micewere allowed to acclimate to the BSL-2 level animal housing facility atthe University of Iowa (Iowa City, Iowa) for at least seven days, priorto their inclusion in this study. All animal work described herein wasapproved by and conducted in accordance with the recommendations ofAnimal Care and Use Committee at the University of Iowa (IACUC #1205097). At DO, 8-12 week old mice were anesthetized with isoflurane,abdominal skin was carefully shaved with an Accu-Edge microtome blade(Sakura-Finnetek, Torrance, Calif.) and exposed skin was cleansed bywiping with an alcohol prep pad (Covidien, Mansfield, Mass.). Forinoculum preparation, a USA 300 MRSA strain (AH1263) or its deletionmutant (AH1292) were grown in TSB medium overnight at 37° C. in ashaking incubator set to 200 rpm. Log-phase bacteria were obtained aftera 2 hr subculture of a 1:100 dilution of the overnight culture in TSB.Bacterial cells were pelleted and resuspended in DPBS to a concentrationof 1×10⁸ CFUs/45 μL. 50 μL inoculum suspensions containing 1×10⁸ CFUsand either 224C-F2 (5 μg, or 50 μg diluted in DMSO) or DMSO alone wereinjected to intradermally into abdominal skin using 0.3 mL/31 gaugeinsulin syringe (BD, Franklin Lakes, N.J.). Infectious dose wasconfirmed by plating serial dilutions of inoculum on TSA and countingensuing colonies after overnight culture. Baseline body weights of micewere measured before infection and every day thereafter for a period of7 days. For determination of lesion size, digital photos of skin lesionswere taken daily with a Canon Rebel Powershot (ELPH 330 HS) and analyzedvia ImageJ software (National Institutes of Health Research ServicesBranch, Bethesda, Md., USA).

224C-F2 Inhibits S. aureus Quorum Sensing Across the Diversity of agrAlleles.

A number of in vitro assays were employed to guide fractionation of thenatural product composition and to evaluate efficacy in blocking S.aureus quorum sensing mediated virulence. Growth inhibitory impact ofthe extracts was assessed with traditional static MIC assays (Table 2);growth inhibition was also tracked in the fluorescent reporter assaysfor agr activity (FIG. 3).

Table 2. Growth and biofilm inhibition studies. Minimum inhibitoryconcentrations (MIC) were determined for extracts 224, 224C, 224C-F2 andcontrol antibiotics (Ampicillin and Kanamycin) against Staphylococcusaureus strains. Minimum biofilm inhibiting concentration (MBIC)determination is also presented, and compared to control extract220D-F2. All MIC and MBIC values are represented in μg mL⁻¹.

Test Agent (μg mL⁻¹) 224C- 220D- Strain ID MIC 224 224C F2 Amp Kan F2AH430 MIC₅₀ 64 64 64 0.0625 2 — MIC₉₀ ND ND ND 0.125 4 — AH1677 MIC₅₀ 3216 64 ND ND — MIC₉₀ ND ND 256 ND ND — AH1747 MIC₅₀ 128  16 8 ND 1 —MIC₉₀ ND ND 256 ND 2 — AH1872 MIC₅₀ 16 64 16 4 1 — MIC₉₀ ND ND 128 8 4 —NRS385 MIC₅₀ 16 16 16 ND ND — MIC₉₀ ND ND 128 ND ND — UAMS-1 MIC₅₀ 32 6432 ND 2 128 MIC₉₀ ND ND ND ND 4 ND MBIC₅₀ 200  100  200 — — 12.5 MBIC₉₀ND ND 400 — — 100224C-F2 Blocks S. aureus Damage to Human Keratinocytes

In addition to monitoring the activity of each agr allele and detectingspecific downstream products (e.g. α-hemolysin and δ-toxin), virulenceimpact data was captured on other exotoxins that could be producedthrough this system. To do this, HaCaT cells were exposed to thesterile-filtered supernatants of treated and control cultures. Thedifference in cytotoxicity as detected by LDH assay was clear (p<0.001)for all extracts (224, 224C, and 224C-F2) in comparison to control, andthis was evident at doses as low as 0.25 μg mL⁻¹ (FIG. 5A). Followingexposure to supernatants (14% v/v for 3 hrs) or staurosporine (7.1 μMfor 3 hrs), HaCaT cells were imaged by fluorescent microscopy to examinecell integrity. Images of the HaCaT cells following exposure to thesupernatants reaffirmed the lack of exotoxins in the supernatants in224C-F2 treated cultures.

Repeated Exposure to 224C-F2 does not Lead to Resistance

Antibiotic resistance is a major concern in any anti-infective drugdiscovery initiative. Here, it is hypothesized that targeting bacterialvirulence with a multi-component botanical therapy—potentiallycontaining multiple actives acting on multiple targets—would not be verylikely to generate resistance. As reporter strains can lose theireffectiveness in tracking activity over multiple passaging days (e.g.due to loss of the plasmid), a method was designed for tracking thequorum quenching efficacy of the composition (224C-F2). This wasachieved through use of a high toxin output strain (NRS385) thatconsistently produces high levels of δ-toxin in the supernatant.Bacterial growth was monitored by OD600 and δ-toxin was quantified byHPLC. Data for total peak area measured by HPLC (FIG. 6A) and areaadjusted for slight differences in daily OD (FIG. 6B) both reflectsignificant differences between the levels of δ-toxin produced by thetreated versus control cultures for 15 days of passaging. Moreover, notrends in the shift of this observation towards resistance were noted.

224C-F2 is Nontoxic to HaCaT Cells and Mouse Skin

To investigate the potential for cytotoxic or irritant effects of C.sativa leaf extracts, immortalized human keratinocyte cells were treatedwith up to 512 μg mL⁻¹ of each extract. In all cases (224, 224C,224C-F2), cytotoxicity (>30%) was only observed at doses at 8-10 timesgreater than the dose range necessary for quorum quenching activity, andwhich also corresponded with the rise in toxicity of vehicle treatmentalone (DMSO), with no significant difference in cytotoxicity between thevehicle and extracts (FIG. 7). With regards to the potential forirritant or necrotic effects on murine skin, mice were injectedintradermally with either 5 μg or 50 μg and monitored for any visiblechanges in the skin morphology and weight loss. No changes were notedany day at up to 6 days of post-injection follow-up.

224C-F2 Attenuates MRSA-induced Illness in an in vivo Skin InfectionModel

The agr quorum sensing system controls staphylococcal virulence factorexpression and is required for necrotic skin lesion formation followingcutaneous challenge. Having demonstrated the quorum sensing inhibitingactivity of 224C-F2 in vitro (FIGS. 3-6), the efficacy of thiscomposition was assessed in a mouse model of S. aureus skin infection.When delivered at the time of infection, 224C-F2 decreased the area ofresultant ulcers in a dose-dependent manner (FIGS. 8A). In addition,224C-F2 administration significantly attenuated infection-inducedmorbidity (assessed by weight loss) compared to vehicle treated controls(FIG. 8B). Importantly, mice receiving intradermal injection of 224C-F2alone did not exhibit any overt signs of dermal irritation or clinicalillness e.g., weight loss, malaise, hunching, coat ruffling (FIG. 7).Together these data corroborate the in vitro findings and suggest that224C-F2 impairs MRSA pathogenesis without manifesting local or systemictoxicity.

224C-F2 Activity Against Propionibacterium acnes

The extract 224C-F2 was tested for growth inhibitory activity againsttwo strains of Propionibacterium acnes. Clindamycin was used as thepositive control. The MIC, or minimum inhibitory concentration forgrowth is represented at MIC50 (or at 50% inhibition of the growthcontrol) or MIC90 for 90% inhibition. Likewise, the MBEC, or minimumbiofilm eradicating concentration, is represented at 50% (MBEC50) and90% (MBEC90) of the mock vehicle control (DMSO or water).

Test Agent (μg mL⁻¹) Species Strain ID MIC 224C-F2 ClinPropionibacterium HL005PA2 MIC₅₀ 128 0.0625 acnes MIC₉₀ ND 0.25Propionibacterium ATCC6919 MIC₅₀ 256 0.125 acnes MIC₉₀ 256 0.125

In addition to testing 224C-F2, other crude extracts of Castanea sativawere also tested against P. acnes strain ATCC 6919, and alsodemonstrated activity:

Extraction Extract Test Agent (μg mL⁻¹) Plant part solvent ID MICExtract Clin Leaves MeOH 224 MIC₅₀ 256 0.125 MIC₉₀ — 0.125 Leaves withgall MeOH 226 MIC₅₀ 256 0.125 structures MIC₉₀ — 0.125

These studies demonstrate that extracts of Castanea sativa leavesexhibit growth inhibitory activity against Propionibacterium acnes andcould hold potential utility in formulations for the treatment of acneflares.

Chemical Characterization of 224C-F2

The percent yield of extract from the dry leaves was 43.98% for extract224, 2.716% for 224C and 1.155% for 224C-F2 (FIG. 2). LC-FTMS analysisof 224C-F2 revealed the presence of at least 94 compounds (Table 4).

TABLE 4 Mass spectrometry (m/z) analysis of 224C-F2. The correspondingchromatogram is reported in FIG. 9; putative structures in FIG. 10 and11. Retention Peak Time Relative Formula m/z* No. (min) Abundance (Δppm) ([M − H]⁻ in bold) MS² UV 1 6.21 0.99 C₁₄H₉O₁₀ 337.02044, 293.2077220, 270 (0.950) 675.05002 2 6.85 0.39 C₁₄H₁₁O₁₀ 339.0354 169.104  220,270 (0.586) 3 9.17 0.12 C₁₄H₉O₈ 305.03035, 179.1553 ND (1.993) 611.068044 9.82 0.24 C₁₄H₉O₉ 321.02541, 169.1691 215, 280 (2.347) 643.05813 510.05 0.32 C₃₁H₁₇O₁₅N 321.02581, 599.13610, 215, 280 (0.313) 579.15259,626.17633 611.10667, 643.06011 6 10.49 0.36 C₈H₇O₅ 183.02972, 124.08116,215, 275 (2.031) 367.06658 168.05430, 183.09955 7 10.94 0.14 C₁₄H₉O₉321.02533, 169.1772 ND (2.097) 643.05801 8 11.18 0.16 C₁₈H₁₅O₈ 33.06169,179.1279 215, 325 (2.138) 359.07746 9 11.74 0.22 C₂₁H₂₅O₈ 405.15626,225.224  ND (1.898) 451.23452, 521.24023 10 12.02 0.19 C₂₅H₃₉O₇269.13963, 407.24461, ND (1.337) 451.27073 433.23288 11 12.39 1.24C₂₈H₁₁O₁₆ 603.0069 465.2773 255, 365 (2.757) 12 13.18 0.33 C₁₅H₁₁O₉335.04137, 183.209  ND (1.537) 671.08973 13 13.36 0.32 C₂₉H₃₃O₁₀421.15146, 491.18281, ND (2.420) 541.20923 523.30375 14 13.68 0.33C₁₅H₁₁O₉ 335.04131, 183.1594 215, 280, (1.358) 671.09010 305 (s) 1514.13 0.33 C₁₂H₁₉O₅ 243.12412, 225.1935 ND (1.329) 487.25602 16 14.470.30 C₁₂H₁₉O₅ 243.12411, 181.17663, ND (1.288) 487.25527 183.17220,199.20072, 225.24659 17 14.71 0.26 C₂₃H₂₃O₅N₄ 435.1674 259.15392, ND(−0.076) 388.97603 18 14.90 0.54 C₃₃H₁₃O₁₃N 391.14110, 479.4478 220, 255(0.081) 631.03874 (s), 320 19 15.44 0.32 C₂₀H₃₁O₈ 399.2033 381.33071, ND(2.101) 355.30539, 337.33447 20 16.26 0.25 C₃₀H₃₉O₈ 527.2661 263.20418,ND (2.046) 459.45849 21 16.63 0.16 C₅₉H₄₁O 405.15614, 613.37801, ND(−1.084) 765.31542 617.27520 22 16.84 0.21 C₂₅H₂₃O₁₃ 599.1065 255.10369,ND (1.179) 284.12921, 285.12352, 327.22129, 471.26204 23 16.97 0.17C₁₅H₉O₆ 285.04071, 175.11613, ND (0.873) 571.08859 199.13418, 241.1428124 17.39 1.15 C₁₅H₉O₇ 301.03595, 151.08622, 215, 255, (1.907) 603.08092,179.07130 370 905.12920 25 17.88 2.08 C₁₈H₃₁O₅ 327.21816, 211.20899,220, 280, (1.414) 655.44500 229.25489, 330 291.32982 26 18.50 0.40C₂₆H₃₉O₆ 447.27607, 367.44245, ND (1.918) 493.28185, 385.41613,895.56483 401.41446, 429.40811 27 18.97 0.08 C₂₅H₂₃O₁₃ 519.33352,471.23936, ND (1.461) 531.11519, 489.30998 564.33918 28 19.81 1.90C₄₃H₆₅O₂₄ 329.23393, 635.3972 220, 270, (−0.265) 635.14327, 315965.38687 29 20.33 1.31 C₅₀H₁₉O 635.143  285.15142, ND (−1.840)489.24712, 575.28563 30 20.79 0.50 C₁₈H₃₁O₅ 327.21830, 171.19486, ND(1.842) 655.44361 309.30290 31 21.45 0.34 C₃₉H₅₉O₈ 327.21808, 611.57685,220, 345 (34.188); 635.14199, 637.50026 C₃₈H₅₅O₉ 655.44397 (89.700) 3222.15 0.30 C₃₅H₅₉O₆ 287.22306, 515.51088, 220, 365 (39.112) 327.21853,531.44821, 419.16528, 557.36796 575.45419, 661.36183, 755.16549 33 22.760.16 C₂₇H₂₃O₁₈ 327.21797, 285.14949, 220, 315 (80.837) 635.1404489.26353 34 23.54 0.65 C₃₉H₃₁O₁₅ 739.1677 285.14592, 220, 270, (1.118)453.23143 315 35 24.11 2.67 C₅₇H₂₃O₂ 739.16945, 285.13965, 220, 315(−1.263) 785.17519 453.25508, 575.28574, 593.30977 36 24.70 2.65C₂₇H₄₁O₆ 461.29190, 399.45155, ND (2.250) 507.29838, 415.44332,923.59481 443.44095 37 25.61 0.90 C₅₇H₂₃O₂ 739.16923, 285.14029, 220,315 (−1.520) 785.17625 453.24568, 575.300080, 593.27713 38 26.00 0.84C₅₅H₂₁ON₃ 739.16895, 285.14254, 220, 315 (−0.163) 785.17576 453.25317,575.30248, 593.29857 39 27.18 0.72 C₁₇H₁₁O₈ 343.04651, 328.2618 225, 370(3.317); 687.10014 C₂₀H₁₁O₄N₂ (75.038) 40 27.57 0.84 C₄₀H₂₇O₁₁N₄739.1683 285.15812, ND (0.026) 453.24020, 575.26598, 593.28341 41 28.450.68 C₃₄H₂₉O₁₅ 677.15166, 284.13571, 220, 310 (0.822) 723.15962557.27766, 617.28692 42 29.30 1.43 C₃₁H₄₉O₆ 517.31804, 437.46551, ND(−68.377) 563.32400, 455.47822 723.15771, 797.17555, 1035.64788 43 30.281.86 C₃₀H₄₅O₇ 517.31837, 437.46548, ND (2.500) 563.32368, 455.47972,1035.64687 499.50877 44 32.74 0.26 C₃₄H₂₉O₁₅ 677.1506 285.14940, ND(−1.423) 531.27599, 617.30259 45 33.59 0.31 C₃₄H₂₉O₁₅ 547.32815,285.1559, ND (−1.112) 677.15045 531.29170 46 35.37 1.64 C₅₇H₂₃O₂N₃781.17897, 285.14510, 225, 310 (−0.557) 827.18606 495.29220, 617.28374,635.31959 47 38.38 3.13 C₅₇H₂₃O₂N₃ 781.17980, 285.14324, 220, 315(0.288) 827.18720 495.26366, 635.32730 48 40.35 1.45 C₅₉H₂₅O₃ 445.29698,285.14614, 220, 285, (−0.676) 491.30248, 496.26557, 310 781.18038,635.31187 827.18746, 1227.49773 49 41.60 1.07 C₅₉H₂₅O₃ 533.34940,285.12660, 220, 305 (−1.201) 781.18002, 495.27804, 827.18679 635.3151750 43.97 1.20 C₄₁H₃₃O₁₆ 781.1779 285.13183, 220, 295 (0.617) 495.28281,635.30008 51 47.42 5.96 C₃₀H₄₇O₅ 487.34398, 469.4979 ND (2.221)533.35098, 975.70011 52 48.55 0.48 C₃₂H₅₁O₇ 547.36365, 529.51760, ND(−0.543) 593.36950 529.51407 53 49.73 0.93 C₃₁H₄₉O₇ 533.3478 435.49468,ND (−1.063) 486.52688, 515.46714 54 51.40 0.67 C₃₀H₄₇O₅ 487.34265,485.22061, ND (−0.508) 533.34807 486.02828, 487.97113 55 52.42 4.11C₃₀H₄₇O₅ 487.34400, 469.499  225, 270 (2.262) 533.35101, 975.70046 5654.43 0.97 C₃₁H₄₉O₈ 531.33301, 489.57421, ND (2.418) 549.34462 531.4158357 56.01 3.84 C₃₀H₄₇O₅ 487.34406, 441.51916, ND (2.385) 533.35100,469.49007 975.70018 58 57.15 4.36 C₅₉H₂₅O₃N₃ 823.18973, 285.13804, 225,315 (−0.498) 869.19736 677.32656 59 58.72 2.88 C₅₉H₂₅O₃N₃ 823.18959,285.14787, 220, 315 (−0.668) 869.19629 677.31854 60 61.17 6.80 C₃₀H₄₇O₆503.33900, 471.4702 ND (2.359) 549.34625, 1007.69059 61 62.79 2.56C₅₉H₂₅O₃N₃ 823.18949, 285.13196, 220, 315 (−0.790) 869.19646 677.3243262 64.42 3.63 C₅₉H₂₅O₃N₃ 823.1893 285.12941, 220, 315 (−1.020) 677.3176863 66.55 3.09 C₃₀H₄₇O₆ 503.33871, 319.32486, ND (1.783) 549.34568,401.40810, 1007.69071 471.48019 64 79.78 2.91 C₃₀H₄₅O₅ 485.32867,423.49110, ND (2.930) 531.33501, 467.49405 971.66969 65 81.93 1.98C₃₀H₄₉O₅ 489.35929, 471.5105 ND (1.517) 535.36558, 979.72844 66 86.292.25 C₂₇H₄₁O₅ 445.29464, 383.45557, ND (1.151) 491.30202, 427.42744891.60136 67 89.69 0.13 C₃₁H₄₉O₇ 533.348  487.4407 ND (−0.744) 68 90.420.46 C₃₁H₅₁O₆ 519.3685 415.49682, ND (−1.179) 487.52239 69 91.30 0.92C₃₉H₅₃O₇ 633.37985, 179.08913, 225, 295, (0.273) 679.38594 454.54671,305 590.61801 70 92.33 1.20 C₃₂H₄₉O₆ 529.35452, 469.5822 ND (1.998)575.36114, 1059.72276 71 92.99 0.51 C₃₂H₄₉O₆ 529.35467, 469.6057 ND(2.281) 575.36044 72 93.19 0.31 C₂₇H₄₃O₄ 431.31834, ND ND (3.842)477.32329, 529.35529 73 93.74 0.16 C₄₀H₅₅O₉ 547.36537, 619.46845, ND(0.447) 679.38546 661.52463 74 94.38 1.26 C₅₃H₉₉O₁₃ 471.35048,471.52443, ND (1.032) 517.35526, 925.86052 943.71008 75 94.85 0.69C₃₉H₅₃O₇ 633.3816 470.54105, ND (2.957) 514.52351, 590.59043 76 95.270.47 C₃₂H₄₉O₆ 529.35483, 469.5901 ND (2.583) 575.36059 77 95.52 0.18C₃₂H₄₉O₇ 545.34949, 485.4473 ND (2.022) 591.35525 78 95.80 0.40 C₃₃H₄₅ON471.35008, ND ND (−1.238) 517.35387, 943.70544 79 96.74 0.82 C₃₂H₄₉O₇545.34956, 453.49263, ND (2.169) 591.35536 485.45412, 513.44457 80 97.000.25 C₃₁H₄₉O₇ 485.32855, 489.44583, ND (1.487) 533.34917, 513.21950591.35511 81 97.34 0.29 C₃₂H₄₉O₇ 545.34923, 485.4417 ND (1.564)591.35526 82 97.56 0.44 C₃₃H₄₅ON 471.34893, 453.5104 ND (−3.677)517.35439 83 98.10 0.39 C₄₈H₅₉O₁₀ 795.4106 633.6017 225, 300, (−0.982)325 84 98.33 0.23 C₄₈H₅₉O₁₀ 485.32931, 633.5975 ND (0.351) 531.33456,795.41162 85 98.81 0.56 C₂₀H₃₉O₇ 391.2714 371.2172 ND (3.152) 86 99.530.57 C₃₉H₅₇O₆ 475.30817, 179.11584, ND (1.685) 533.34947, 451.48783,621.41711 577.68246, 603.59836 87 100.29 1.85 C₂₇H₄₁O₄ 429.30177,367.42174, ND (1.717) 475.30774 411.43779 88 101.24 0.21 C₃₀H₄₉O₄473.36460, ND ND (2.190) 519.36979 89 102.05 1.32 C₃₀H₄₇O₄ 471.34907,367.41415, ND (2.306) 517.35508, 409.51672, 943.70971 453.51813 90102.67 0.10 C₃₉H₅₃O₆ 455.31789, 497.49844, ND (0.028) 501.32245,573.61572 617.38478 91 103.00 0.17 C₃₀H₄₇O₅ 487.34309, 469.4995 ND(0.394) 533.34826, 975.69540 92 103.59 0.04 C₂₉H₄₅O₄ 457.33254,395.50910, ND (0.452) 503.33759, 439.45901 915.67143 93 103.90 0.20C₃₀H₄₇O₄ 471.34849, 413.50334, ND (1.075) 517.35330, 453.50566 943.7032494 104.39 0.06 C₃₀H₄₇O₅ 487.34298, 455.4734 225, 290, (0.169) 533.34853,435 975.69146

The greatest quorum quenching effects of 224C-F2 were observed in theretention time region of 21-49 min (FIG. 9), suggesting the presence ofseveral distinct quorum quenching compounds (data not shown).Specifically, there are 22 compounds found in this region, 10 presentat >1% relative abundance. These correspond to peak numbers, predictedformulas, and relative abundances of: 35 C₅₇H₂₄O₂ (2.67%), 36 C₂₇H₅₀O₆(2.65%), 42 C₃₁H₅₀O₆ (1.43%), 43 C₃₀H₄₆O₇ (1.86%), 46 and 47 C₅₇H₂₃O₂N₃(1.64% and 3.13%, respectively), 48 and 49 C₅₉H₂₅O₃ (1.45 and 1.07%,respectively), 50 C₄₁H₃₃O₁₆ (1.20%) and 51 C₃₀H₄₇O₅ (5.96%). Putativestructures for 7 peaks were determined to be pentacyclic triterpenes(specifically, oleanene and ursene derivatives) based on accurate massanalysis, fragmentation patterns, and comparison with natural productdatabases (FIG. 10), and these collectively represent 16.37% in relativeabundance. Of note, while present at relative abundance levels of <1%each, the putative structures of gallotannins (32, 33, 34) andellagitannins (39) were also identified in the most active region of224C-F2.

224C-F2 was also examined by HPLC-DAD and LC-FTMS for the presence of 5compounds reported to be found in crude C. sativa leaf extracts, and itwas determined that 224C-F2 does not contain chlorogenic acid, ellagicacid, hyperoside, isoquercitrin, or rutin.

TABLE 3 Inhibition of S. aureus quorum sensing by chestnut leaf extractsas detected by agr reporter strains. Test Agent (μg mL⁻¹) Strain ID agrgroup IC 224 224C 224C-F2 AH1677 I IC₅₀ 100 50 25 IC₉₀ ND ND 100 AH430II IC₅₀ 25 50 12.5 IC₉₀ ND ND 100 AH1747 III IC₅₀ 25 12.5 1.56 IC₉₀ ND100 12.5 AH1872 IV IC₅₀ 100 100 25 IC₉₀ ND ND 100

A slightly higher level of growth inhibition was observed in the staticMIC assays over that observed in the super-aerated reporter assay, butin all reporter strains, the MIC remained >100 μg mL⁻¹ for 224C-F2. Nobiofilm inhibitory or promoting activity of the extracts was noted.

Quorum quenching effects for 224C-F2 were observed at ICso values of1.56-25 μg mL⁻¹, depending upon the strain tested (Table 3). The mostpotent quorum quenching activity was observed for agr III (IC₅₀ of 1.56μg mL⁻¹), and the least for agr IV (IC₅₀ of 25 μg mL⁻¹). Significantinhibition of agr was observed for all agr alleles at sub-inhibitoryconcentrations for growth, indicating that the quorum-quenching activityis due to specific interference with agr, and not simply the result of afalse positive due to growth inhibition.

To verify the observed quorum quenching activity, downstreamtranslational products of the quorum sensing system were assessed. HPLCquantification of δ-toxin (FIG. 4A) from the supernatant of a heavyproducer of exotoxins (NRS385, a USA 500, agr I, HA-MRSA isolate)revealed significant reduction (p<0.01) in production of δ-toxin in224C-F2 treated cultures at doses as low as 0.25 μg mL⁻¹ (FIG. 4B).

To verify the block in production of additional exotoxins, cultures ofstrain LAC (AH1263, a USA300, agr I, CA-MRSA isolate) and its isogenicagr (AH1292) and hla (AH1589) mutants were grown in the presence of theextracts and their supernatants were examined in a rabbit red blood celllysis assay. In this assay, the majority of RBC lysis is attributed tothe presence of α-hemolysin in the culture supernatant. The presence ofsome lytic activity in the Ahla vehicle control suggests that someadditional hemolytic activity (˜18%) may be due to additional toxins inthe supernatant, phenol soluble modulins (PSMs), in particular.Treatment of wild type with 224C-F2 resulted in significant (p<0.001)reduction in hemolytic activity in wild type strain at 6.25 μg mL⁻¹, andalmost total loss of hemolytic activity at the concentration of 100 μgmL⁻¹. Treatment of the Ahla mutant demonstrated nearly total loss ofhemolytic activity at 6.25 μg mL⁻¹ (FIG. 4C). Similar to the hemolysisassessment, when USA300 is exposed to increasing doses of all extracts(224, 224C, and 224C-F2), the level of a-hemolysin protein production ismarkedly attenuated, with the most potent activity exhibited by 224C-F2(FIG. 4D).

Sub Fraction Analysis

RP HPLC semi-prep chromatogram of 224C-F2c was performed at 314 nm (SeeFIG. 12). FIG. 13 shows Fractions 7 and 11 (note HPLC vial numbers donot correspond to the fraction numbers). FIGS. 14 and 15 show LC-FTMSESI—for 224C-F2c-SF7 and SF11 respectively. FIG. 16 shows dataindicating inhibition of agr without impacting growth.

Table 4 Shows Data for 224C-F2c-SF7

Retention Putative Time Formula Peak (min) (Δ ppm) [M-H]^(-*) MS-MSfragments 1 57.8 C39H61O7N (0.2) 327.2185, 655.4447 593.5429, 611.5981,637.6499 2 58.08 C39H61O7N (0.2) 327.2185, 575.4564, 593.5785, 611.6195,637.5610 615.4506, 655.4483 3 58.73 C39H31O15 (1.1) 739.1666 ND 4 59.04C39H31O15 (1.7) 739.167 285.1537, 453.2921, 575.3205, 593.3320 5 59.17C22H33O22N2 677.152 285.1352, 531.2750, 617.2969 (0.1)

Table 5 Shows Data for 224C-F2c-SF11

Retention Putative Time Formula Peak (min) (▭ ppm) [M-H]^(-*) MS-MSfragments 1 55.42 C31H51O9 (−1.5) 567.3530 535.5784 2 57.15 C18H33O5(−0.2) 329.2333 211.2389, 229.2811, 311.3833 3 59.80 C30H45O7 (1.5)517.3179 437.5225, 455.5107 4 59.97 C30H45O7 (2.8) 517.3179 437.4781,455.5008 5 60.37 C27H35ON4 (0.5) 431.2813 369.4419, 413.4428 6 60.69C41H33O16 781.1759 285.1579, 495.3045, (−2.0) 635.3421 7 60.93 C41H33O16781.1762 285.1545, 495.2740, (−1.4) 635.3508 8 62.74 C28H45O6 (2.2)477.3233, 431.5300, 459.5256 523.3295, 955.6565 9 63.04 C24H35O5 (1.9)403.2798, 357.4009, 385.4288 449.2562, 807.5076

1. An extract comprising a leaf derived mixture of compounds from aCastanea plant wherein the extracting process comprises the steps of:mixing a leaf with methanol under conditions such that leaf compoundsdissolves in the methanol and removing the methanol providing a methanolderived mixture of compounds, partitioning the methanol derived mixtureof compounds in hexane and water providing a water derived mixture ofcompounds, partitioning the water derived mixture of compounds by mixingthe water with ethyl acetate under conditions such that leaf compoundsdissolve in the ethyl acetate and removing the ethyl acetate providingan ethyl acetate derived mixture of compounds; and purifying the ethylacetate derived mixture of compounds by liquid chromatography throughsilica with a mobile phase comprising hexane and ethyl acetate; whereinthe mobile phase comprises increasing amounts of ethyl acetate, and amobile phase fraction is isolated comprising a leaf derived mixture ofcompounds which does not contain chlorogenic acid, ellagic acid,hyperoside, isoquercitrin, or rutin.
 2. The extract of claim 1, whereinthe mixture of compounds comprises components having the followingformulas and relative abundance, C₃₉H₅₉O₈ or C₃₈H₅₅O₉, or mixture, witha relative abundance of 0.1 to 0.9%; C₃₅H₅₉O₆ with a relative abundanceof 0.1 to 0.9%; C₃₁H₄₉O₆ with a relative abundance of 1.0 to 2.0%;C₃₂H₅₁O₇ with a relative abundance of 0.1 to 0.9%; C₃₀H₄₈O₅ with arelative abundance of 3.0 to 5.0%; C₃₀H₄₈O₆ with a relative abundance of6.0 to 8.0%; and C₃₀H₄₅O₅ with a relative abundance of 2.0 to 4.0%. 3.The extract of claim 3, further comprises additional components ofdifferent compounds having the following formula and relative abundance,C₃₅H₅₉O₆ with a relative abundance of 0.2 to 0.4; C₂₇H₂₃O₁₈ with arelative abundance of 0.05 to 0.30; C₃₉H₃₁O₁₅ with a relative abundanceof 0.50 to 0.80; C₁₇H₁₁O₈ or C₂₀H₁₁O₄N₂, or mixture, with a relativeabundance of 0.5 to 1.0; and C₃₄H₂₉O₁₅ with a relative abundance of 0.2to 0.4%.
 4. The extract of claims 1, wherein the mixture comprises atleast one component from each of the following groups a) to g): a) acompound selected from escigenin tetraacetate (6CI); tetraacetate(7CI,8CI) 16α,21α-epoxy-olean-9(11)-ene-3β,22β,24,28-tetrol;tetraacetate aescigenin; triacetate (8CI) cyclic16,22-acetal-olean-12-ene-3β,16α,21β,22α,28-pentol; and triacetate (8CI)cyclic 22,28-acetal-olean-12-ene-3β,16α,21β,22α,28-pentol, or mixture oftwo, three, four or all; b) a compound selected from stigmastane and(3β,4β,16α,21β,22α)-16,21,22,23,28-pentamethoxy (9CI) olean-12-en-3-ol,or mixture; c) amirinic acid; d) a compound selected from 21-acetateprotoescigenin, 16-acetate protoescigenin, 22-acetate protoescigenin and28-acetate protoescigenin, or mixture of two, three, four or all; e) acompound selected from16,21-epoxy-(3β,4β,16α,21α,22β)-olean-12-ene-3,22,24,28-tetrol (9CI);asiatic acid; arjunolic acid; and isoescigenin, or mixture of two,three, or all; f) a compound selected from camelliagenin E; brahmicacid; sericic acid; belleric acid; and2,3,23,24-tetrahydroxy-(2α,3β)-urs-12-en-28-oic acid, or mixture of two,three, four or all; and g) ouillaic acid.
 5. The extract of claim 4,wherein the mixture comprises at least one component from each of thefollowing groups a) to d): a) a compound selected from stigmastane and(3β,4β,16α,21β,22α)-16,21,22,23,28-pentamethoxy (9CI) olean-12-en-3-olor a mixture; b) a compound selected from 1,3,6-tri-O-galloylglucose;1,2,6-tri-galloyl-β-D-glucose; 1,2,3 -tri-O-gall oylglucose; 1,2,3-tri-O-galloyl-β-D-glucopyranose; 2′,3,5-tri-O-galloyl-D-hamamelose;2-C-[[(3 ,4, 5-trihydroxyb enzoyl)oxy]methyl]-1,5-bis(3 ,4,5-trihydroxybenzoate) D-ribofuranose; kurigalin; and3,4,6-tri-O-galloyl-D-glucose, or mixture of two, three, four, five,six, seven, or all; c) castanoside B; and d) a compound selected from3,4,3′-tri-O-methylellagic acid and 3,3′,4′-tri-O-methylellagic acid, ora mixture.
 6. A pharmaceutical or cosmetic formulation comprising theextract or one or more compounds in the extract of claim 1 and apharmaceutically acceptable excipient or cosmetically acceptableexcipient.
 7. A liquid or gel formulation of claim 6, optionally furthercomprising an antibiotic agent, a topical steroid, an anti-inflammatoryagent, a promoter of skin barrier function, a skin moisturizer orcombinations thereof.
 8. The formulation of claim 6 comprising anenteric coating.
 9. A solid or liquid soap or lotion comprising theextract or one or more compounds in the extract of claim 1 and a fattyacid.
 10. A medical device comprising a coating comprising the extractor one or more compounds in the extract of claim
 1. 11. A wounddressings or wound rinse comprising the extract or one or more compoundsin the extract of claim 1 wherein the wound dress comprises an absorbentpad and optionally an adhesive.
 12. A tampon comprising the extract orone or more compounds in the extract of claim 1 wherein the tamponcomprises an absorbent material.
 13. A method of treating or preventinga bacterial infections comprising administering or contacting a formulacomprising the extract or one or more compounds in the extract as inclaim 1 to a subject in need thereof.
 14. The method of claim 13,wherein the subject is at risk of, exhibiting symptoms of, or diagnosedwith toxic shock syndrome, scalded skin syndrome, abscesses, furuncles,cellulitis, folliculitis, bloodstream infections, medical deviceinfections, pneumonia, osteomyelitis, staphylococcal food poisoning,skin and soft tissue infections, endocarditis, eczema, atopicdermatitis, psoriasis, impetigo, septic arthritis, brain abscess, burnwounds, venous ulcers, diabetic foot ulcers, surgical wounds,post-operation infections, carbuncles, meningitis, bacteremia,necrotizing pneumonia, or necrotizing fasciitis.
 15. The method of claim13, wherein the formula is administered in combination with anantibiotic agent.
 16. A method of treating acne comprising administeringa composition comprising an extract the extract or one or more compoundsin the extract as in claim 1 to a subject at risk of, exhibitingsymptoms of, or diagnosed with abnormal acne.